Resolution of inflammation is essential for tissue homeostasis and represents a promising approach to inflammatory disorders. Here we found that developmental endothelial locus-1 (DEL-1), a secreted protein that inhibits leukocyte–endothelial adhesion and inflammation initiation, also functions as a non-redundant downstream effector in inflammation clearance. In human and mouse periodontitis, waning of inflammation was correlated with DEL-1 upregulation, whereas resolution of experimental periodontitis failed in DEL-1 deficiency. This concept was mechanistically substantiated in acute monosodium-urate-crystal-induced inflammation, where the pro-resolution function of DEL-1 was attributed to effective apoptotic neutrophil clearance (efferocytosis). DEL-1-mediated efferocytosis induced liver X receptor–dependent macrophage reprogramming to a pro-resolving phenotype and was required for optimal production of at least certain specific pro-resolving mediators. Experiments in transgenic mice with cell-specific overexpression of DEL-1 linked its anti-leukocyte-recruitment action to endothelial cell–derived DEL-1 and its efferocytic/pro-resolving action to macrophage-derived DEL-1. Thus, the compartmentalized expression of DEL-1 facilitates distinct homeostatic functions in an appropriate context that can be harnessed therapeutically.
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The data that support the findings of this study are available from the corresponding authors upon reasonable request.
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This work was supported by grants from the European Research Council (DEMETINL to T.C.), the Deutsche Forschungsgemeinschaft (Transregio-SFB 83 to Ü.C. as well as Transregio-SFB 127 and Transregio-SFB 205 to T.C.), and the National Institutes of Health (AI068730, DE024153, DE024716, DE026152, and DE015254 to G.H.). I.K. was supported by the MeDDriveGrant (60.400) from the Technische Universität Dresden Medical Faculty. A.Castrillo was supported by Spanish MINECO SAF2014-56819R and SAF2015-71878-REDT. This work was supported in part by intramural National Institute of Dental and Craniofacial Research, National Institutes of Health funding. We thank R. Naumann (Transgenic Core Facility, Max-Planck Institute for Cell Biology and Genetics Dresden, Germany) for microinjections and B. Gercken and M. Schuster (Institute for Clinical Chemistry and Laboratory Medicine, Faculty of Medicine, Technische Universität Dresden, Dresden, Germany) for technical assistance. Parts of this work are included in the master’s theses of C.D. at the Technische Universität Dresden, Dresden, Germany, and A.F. at the University of Porto, Porto, Portugal.
The authors declare no competing interests.
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Integrated supplementary information
a, Probing pocket depth (PPD) was determined in human patients diagnosed with periodontitis before and after scaling and root planing (SRP). b, The protein concentration of DEL-1 was measured in the supernatant of peritoneal exudates obtained from wild-type (WT) or Del-1KO mice at steady state by ELISA (pg/mg of total protein; n = 4 mice per group). c, Relative mRNA expression of the indicated molecules in gingival tissue from wild-type (WT) or Del1KO mice, which were subjected to LIP for 5 d and were euthanized immediately (5 days ligated; 5DL) or 5 d later with the ligatures removed to enable resolution (5 days ligated and 5 days with ligatures removed; 5DL-5DR). Data were normalized to Gapdh mRNA and are presented as fold change relative to baseline (NL, not ligated), which was set as 1 for each genotype (n = 5 mice per group). d, Wild-type (WT) or Del1KO mice were subjected to LIP for up to 5 or 10 d and ligatures were removed or not at day 5 (NL, not ligated; 5DL, 5 days ligated; 10DL, 10 days ligated; 5DL-5DR, 5 days ligated and 5 days with ligatures removed). Numbers of neutrophils in gingival tissues were assessed by immunostaining (n = 6 mice per group). e–g, Wild-type (WT) and Del1KO mice were subjected to LIP and ligatures were removed on day 5. RvD1 (100 nM) or PBS control was then administered on a daily basis until the day before sacrifice, which was performed on day 10 (that is, total of five doses). e, Bone loss was measured on day 10 (n = 6 mice per group). f,g, The concentrations of IL-17A and TGF-β1 in gingival tissue were assayed by ELISA on day 10 (n = 6 mice per group; pg of cytokines/mg of total protein are shown). a, P < 0.0001, pre-post paired two-way ANOVA with repeated measures by patient. b–g, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, non-significant. b,c, Two-tailed Mann–Whitney U test; d, two-way ANOVA with Sidak’s multiple-comparisons test; e, one-way ANOVA, Sidak’s multiple-comparisons test; f,g, two-tailed Student’s t test. Data are means +/− s.e.m. and are derived from one experiment (b,c) or pooled from two experiments (d–g).
a, Representative FACS plot from a wild-type mouse at 24 h after intraperitoneal MSU crystal injection showing the gating strategy of leukocytes present in peritoneal exudates. R1, neutrophils; R2, monocytes/macrophages; R3, eosinophils. b, Numbers of monocytes/macrophages (Mono+Mac) in peritoneal exudates obtained from wild-type (WT) and Del1KO mice 8 h (n = 11 WT and 13 Del1KO mice) and 24 h (n = 18 WT and 16 Del1KO mice) after MSU crystal–induced inflammation. c,d, Numbers of neutrophils (c) and monocytes/macrophages (Mono+Mac) (d) in peritoneal exudates obtained from wild-type (WT) mice and mice deficient in αL integrin (ItgalKO) 8 h after MSU crystal–induced inflammation (n = 6 WT and 7 ItgalKO mice). e,f, Wild-type mice were injected intravenously with 50 μg of a neutralizing antibody against αM integrin (anti-αM) or isotype control and then were treated with MSU crystals. The numbers of peritoneal neutrophils (e) and monocytes/macrophages (Mono+Mac) (f) were assessed 8 h after MSU injection (n = 6 mice per group). g, Relative mRNA expression of Gpr18, Chemr23 and Fpr2 in peritoneal monocytes/macrophages sorted from wild-type (WT) and Del1KO mice 24 h after MSU-induced inflammation. Relative mRNA expression was normalized against 18S and was set as 1 in wild-type monocytes/macrophages (n = 5 WT and 4 Del1KO mice). h, Wild-type (WT) and Del1KO mice were treated with MSU crystals and RvD1 or control PBS (–). The relative number of apoptotic neutrophils in peritoneal exudates 24 h after treatment is shown. Numbers of apoptotic neutrophils are expressed relative to the WT control (–) group set as 1 (n = 13 mice for the WT and WT+RvD1 groups and n = 12 mice for the Del1KO and Del1KO+RvD1 groups). *P < 0.05, **P < 0.01. NS, non-significant. b, Two-tailed Student’s t test; c–g, two-tailed Mann–Whitney U test; h, one-way ANOVA with Tukey’s multiple-comparisons test. Data are means +/− s.e.m. and are pooled from two experiments (b, 8 h time point and h), from three experiments (b, 24 h time point) or are derived from one experiment (c–g).
Supplementary Figure 3 The effect of DEL-1 on macrophage gene expression at steady state or in the setting of efferocytosis.
(a), Relative mRNA expression of Tgfb1 and Lxra in peritoneal F4/80+CD11b+ macrophages sorted from untreated wild-type (WT) or Del1KO mice (not subjected to MSU crystal–induced inflammation). Relative expression was normalized against 18S and was set as 1 in WT macrophages (n = 6 WT and 9 Del1KO mice). (b,c), Relative mRNA expression of Tgfb1 in wild-type bone marrow-derived macrophages (BMDMs) (n = 6 separate cell isolations) (b) and isolated peritoneal macrophages from untreated wild-type mice (n = 8 separate cell isolations) (c) upon co-culture of either with mouse apoptotic neutrophils for 3 h in the presence of Fc control or DEL-1 Fc (each 500 ng/ml). (d–i), Relative mRNA expression of Lxra (n = 11 separate cell isolations) (d), Lxrb (n = 12 separate cell isolations) (e), Ucp2 (n =12 separate cell isolations) (f), Abca1 (n = 12 separate cell isolations) (g), Tgm2 (n = 6 separate cell isolations) (h), and Rxra (n = 6 separate cell isolations) (i) in wild-type BMDMs upon co-culture with mouse apoptotic neutrophils for 3 h in the presence of Fc control or DEL-1 Fc (each 500 ng/ml). (j), Relative mRNA expression of Tgfb1 and Lxra in wild-type BMDMs treated with Fc control or DEL-1 Fc (500 ng/ml each; n = 6 separate cell isolations for Tgfb1 and 4 separate cell isolations for Lxra). In (b–j), relative expression was set as 1 in cells that were treated with Fc control in each case. Relative mRNA expression was normalized against 18S. *P < 0.05, **P < 0.01. NS, non-significant. (a,b,e,f,h–j), Two-tailed Mann–Whitney U test; (c,d,g), two-tailed Student’s t test. Data are means +/− s.e.m. and are derived from one experiment (a), from one experiment representative of two (b,h–j), or are pooled from two experiments (c–g).
Supplementary Figure 4 The impact of specific sites of DEL-1 and of LXR signaling on the phenotype of efferocytic macrophages.
(a–c), Relative mRNA expression of Tgfb1 (a), Lxra (b), and Lxrb (c) in wild-type bone marrow-derived macrophages (BMDMs) upon co-culture with apoptotic neutrophils for 3 h in the presence of Fc control, DEL-1 that is mutated in the RGD motif (DEL-1[RGE] Fc) or the truncated protein, in which discoidin I–like domains are absent (DEL-1[E1-E3] Fc) (each 500 ng/ml) is shown (n = 6 separate cell isolations in (a,b) and 12 separate cell isolations in c). d, Shown is relative Lxra and Tgfb1 mRNA expression in Itgb3KO BMDMs that were co-cultured with apoptotic neutrophils for 3 h in the presence of Fc control or DEL-1 Fc (each 500 ng/ml) (n = 5 separate cell isolations). In (a–d), gene expression was normalized against 18S; relative expression was set as 1 in BMDMs that were treated with Fc in each case. (e), Relative Tgfb1 mRNA expression in wild-type BMDMs that were treated with DEL-1 Fc and co-cultured with mouse apoptotic neutrophils for 3 h in the presence of an LXR antagonist (1 μM) or vehicle control (DMSO; Ctrl). Relative mRNA expression was normalized against 18S; relative expression was set as 1 in the presence of vehicle control (n = 15 separate cell isolations). (f,g), Relative Tgfb1 mRNA expression in wild-type BMDMs treated with an LXR antagonist (1 μM) or vehicle control (DMSO; Ctrl) (n = 16 separate cell isolations) (f) and untreated BMDMs from wild-type (WT) mice or from mice deficient in both LXRα and LXRβ (Lxra-LxrbDKO) (n = 5 separate cell isolations per genotype) (g). Relative expression was normalized against 18S and was set as 1 in BMDMs treated with vehicle control (Ctrl) (f) or in wild-type BMDMs (g). *P < 0.05. NS, non-significant. (a–c), One-way ANOVA with Dunnett’s multiple-comparisons test; (d,f,g), two-tailed Mann–Whitney U test; e, two-tailed Student’s t test. Data are means +/− s.e.m. and are from one experiment representative of two experiments (a,b), pooled from two (c) or three (e,f) experiments, or derived from one experiment (d,g).
Supplementary Figure 5 Characterization of mice overexpressing DEL-1 or segments thereof in macrophages.
(a,b), Relative mRNA expression of Del1 in isolated peritoneal macrophages and bone marrow-derived macrophages (BMDMs) from untreated (not subjected to MSU crystal–induced inflammation) mice that overexpress in macrophages full-length DEL-1 (CD68-Del1 mice) (a) or the truncated version of DEL-1 that lacks the discoidin I–like domains (CD68-Del1-[E1-E3] mice) (b) and from their respective untreated wild-type (WT) mice. Relative expression was normalized against 18S and was set as 1 in macrophages from WT mice in each case (a, n = 6 WT and 8 CD68-Del1 mice; (b), n = 6 mice per group). (c,d), Numbers of neutrophils (c) and relative number of apoptotic neutrophils (d) in peritoneal exudates obtained from CD68-Del1-[E1-E3] mice and wild-type (WT) mice 24 h after MSU crystal–induced inflammation. Numbers of apoptotic neutrophils are expressed relative to the WT group, set as 1 (n = 8 mice per group). **P < 0.01, NS, non-significant. (a,b,d), Two-tailed Mann–Whitney U test; c, two-tailed Student’s t test. Data are means +/− s.e.m. and are pooled from two (a) or three (b–d) experiments.
Supplementary Figure 6 The role of integrins on DEL-1-dependent efferocytosis and liposome quality control.
(a,b), Isolated peritoneal macrophages from untreated wild-type (WT) or CD68-Del1 mice were co-cultured for 30 min with apoptotic neutrophils that were prestained with BCECF-AM in the presence of IgG2b (a) or IgG2a (b) control antibody, neutralizing antibody against Mac-1 integrin or against ICAM-1 (a), or antibody against LFA-1 integrin (b) (each antibody 10 μg/ml). Relative efferocytosis index is shown and was calculated as the ratio of macrophages that have phagocytosed apoptotic material to the total number of macrophages. Data are expressed relative to the control group (a, WT-IgG2b; b, WT-IgG2a), set as 1 (n = 5 separate cell isolations per genotype). (c), Representative TLC of liposomes composed of PC/Chol. or PC/Chol./PS stained with primuline. Lanes 1–3 show lipid standards; PC (lane 1), PS (lane 2), cholesterol (Chol., lane 3). (d), Zeta potential measurements of PC/Chol. and PC/Chol./PS liposomes are presented (n = 3 separate preparations). **P < 0.01, NS, non-significant. (a,b), One-way ANOVA with Tukey’s multiple-comparisons test. Data are means +/− s.e.m. and are from three experiments (a,b,d). The experiment in c is representative of three.
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