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Targeting SLC7A11 improves efferocytosis by dendritic cells and wound healing in diabetes

Abstract

Chronic non-healing wounds are a major complication of diabetes, which affects 1 in 10 people worldwide. Dying cells in the wound perpetuate the inflammation and contribute to dysregulated tissue repair1,2,3. Here we reveal that the membrane transporter SLC7A11 acts as a molecular brake on efferocytosis, the process by which dying cells are removed, and that inhibiting SLC7A11 function can accelerate wound healing. Transcriptomics of efferocytic dendritic cells in mouse identified upregulation of several SLC7 gene family members. In further analyses, pharmacological inhibition of SLC7A11, or deletion or knockdown of Slc7a11 using small interfering RNA enhanced efferocytosis in dendritic cells. Slc7a11 was highly expressed in dendritic cells in skin, and single-cell RNA sequencing of inflamed skin showed that Slc7a11 was upregulated in innate immune cells. In a mouse model of excisional skin wounding, inhibition or loss of SLC7A11 expression accelerated healing dynamics and reduced the apoptotic cell load in the wound. Mechanistic studies revealed a link between SLC7A11, glucose homeostasis and diabetes. SLC7A11-deficient dendritic cells were dependent on aerobic glycolysis using glucose derived from glycogen stores for increased efferocytosis; also, transcriptomics of efferocytic SLC7A11-deficient dendritic cells identified increased expression of genes linked to gluconeogenesis and diabetes. Further, Slc7a11 expression was higher in the wounds of diabetes-prone db/db mice, and targeting SLC7A11 accelerated their wound healing. The faster healing was also linked to the release of the TGFβ family member GDF15 from efferocytic dendritic cells. In sum, SLC7A11 is a negative regulator of efferocytosis, and removing this brake improves wound healing, with important implications for wound management in diabetes.

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Fig. 1: SLC7A11 acts as a brake on DC engulfment of apoptotic cells.
Fig. 2: Accelerated skin wound healing in the context of SLC7A11 blockade.
Fig. 3: Glycolysis and glycogen reserves fuel enhanced efferocytosis.
Fig. 4: SLC7A11 inhibition and GDF15 promote wound healing in skin of diabetic mice.

Data availability

All of the sequencing data associated with this work have been deposited in public data bases. Any other related information is available upon request. Source data are provided with this paper.

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Acknowledgements

We thank members of the Ravichandran laboratory and B. Wiernicki for discussions and input; T. L. Aaes for Triwise analysis, and the VIB-Transgenic Core, VIB-Flow Cytometry Core, VIB-Bioimaging Core and VIB-Nucleomics Core; and the VBCF Metabolomics Facility, Vienna, for metabolomics analysis. K.S.R. is supported by FWO (Odysseus grant G0F5716N, EOS DECODE 30837538), Special Research Fund UGent (iBOF BOF20/IBF/037), European Research Council (ERC) (grant agreement no. 835243), grants from NHLBI (P01HL120840), NIAID (R01AI159551), NIGMS (R35GM122542), and the Center for Cell Clearance/University of Virginia School of Medicine. G.v.L. is supported by Foundation against Cancer (STK 2014-142 and STK 2018-093) and FWO (G020216N). S.M. is supported by postdoctoral Marie Skłodowska-Curie Actions individual fellowship (800446) from the European Commission, Horizon 2020 Research and Innovation Framework Program, and E.H. is supported by an FWO postdoctoral fellowship and FWO research grant. Additional support was received through the FWO Postdoctoral Fellowship (1227220N to P.M.). Mouse images in Figs. 1a,g, 2b,g, 3a, 4g,j and Extended Data Fig. 7d (mitochondria and pyruvate transporter) created with BioRender.com. We thank Dr. Sato (Niigata University, Japan) for the Slc7a11 knockout mice.

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Authors and Affiliations

Authors

Contributions

S.M. and K.S.R. designed all experiments and wrote the manuscript. S.M. performed most experiments. E.H. provided conceptual advice and help for skin wound healing experiments. P.M. helped design and assisted with the metabolism-related experiments, B.N.K. assisted with histology studies, and H.K.L.D.C. assisted with immunoblotting experiments. J.P. assisted with bioinformatic analysis. K.L., R.V.d.C., P.J., G.v.L., D.E. and A.M. provided mice, technical advice and input on the manuscript.

Corresponding authors

Correspondence to Sophia Maschalidi or Kodi S. Ravichandran.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Analyzing amino acid transporters during dendritic cell efferocytosis.

a, SLC programs modulated in dendritic cells during efferocytosis of apoptotic cells compared to sterile phagocytosis. RNAseq was performed on primary BMDCs after engulfment apoptotic human Jurkat cells or beads. The heatmap illustrates SLCs upregulated and downregulated during dendritic cell efferocytosis. b, Uptake of CypHer5E- labelled apoptotic Jurkat cells by dendritic cells (BMDC) silenced for Slc7a1 expression (n = 2 per condition). c, Slc7a5 siRNA targeting in dendritic cells does not significantly affect apoptotic cell uptake as assessed by Incucyte Live-cell imaging (n = 4 per group). d, Efferocytosis of TAMRA-labelled apoptotic Jurkat cells by dendritic cells treated with different concentrations of the Slc7a5 inhibitor, JPH-203 (n = 2 per condition). e, Kinetics of efferocytosis by BMDC treated with 3 μM JPH-203 (n = 3 per condition). f, Measurement of degradation of TAMRA (pH insensitive) and Cell Trace Violet (pH sensitive) co-labelled apoptotic targets after efferocytosis by Slc7a11 KO and WT dendritic cells (n = 4 per group). Dendritic cells were incubated with apoptotic targets at a 1:5 phagocyte:target ratio for four hours. Floating bars show minimum to maximum values with all independent replicates, line denotes mean. g, Kinetics of efferocytosis by Slc7a11 WT and Slc7a11 KO bone marrow-derived macrophages with or without erastin treatment (n = 4 per group). ns: not significant; via One-way ANOVA with Tukey’s multiple comparisons test. h, Efferocytosis by peritoneal macrophages after Slc7a11 inhibition via erastin. (n = 8 per condition; P = 0.19, with paired, two-tailed t-test). c, e, g, All live-cell imaging data are expressed as mean ± SEM.

Source Data

Extended Data Fig. 2 Ferroptosis inducer and DC efferocytosis.

a, Phagocytosis of E. coli bioparticles by dendritic cells measured at different time points with or without erastin treatment. Data are expressed as mean ± SD with n = 2 per condition. b, Ferroptosis inducer ML-162 does not enhance efferocytosis by dendritic cells. Live-cell imaging data are expressed as mean ± SEM (n = 3, DMSO and CytoD; n = 4, ML-162; n = 6, Erastin). c, Kinetics of efferocytosis by dendritic cells treated with antioxidant, Ferrostatin-1, erastin alone, or erastin + Ferrostatin-1 (n = 4 per condition, data are representative of two independent experiments). d, Measurement of glutathione levels in dendritic cells treated with erastin (n = 3 per condition, *** P < 0.001; via unpaired two-tailed t-test). e, Assessment of erastin drug cytotoxicity in dendritic cells by measuring Sytox Green fluorescence. Data are expressed as mean ± SD with n = 2 per condition. f, Measurement of lipid peroxidation and ROS (via C11-BODIPY and dihydrorhodamine 123 probes, respectively) in dendritic cells treated with ferroptosis inducers (n = 3 per condition). Results are expressed as fold change (FC). g, Direct comparison of kinetics of efferocytosis by dendritic cells treated with glutamate or erastin (n = 4, Glutamate and Erastin; n = 8, DMSO). h, Kinetics of efferocytosis after NAC, erastin alone or erastin + NAC treatment. DMSO was used as a vehicle control (n = 7, per condition). All live-cell imaging data (b, c, g, h) are expressed as mean ± SEM, * P < 0.05 *** P < 0.001; ****P < 0.0001; One-way ANOVA with Tukey’s multiple comparisons test. ns: non-significant.

Source Data

Extended Data Fig. 3 Contribution of GSH and ROS in the context of enhanced efferocytosis by Slc7a11KO/inhibition.

a, b, GSH supplementation or depletion does not affect the enhanced efferocytosis by DCs lacking Slc7a11 or treated with erastin. WT (n = 6) and Slc7a11 KO (n = 6) dendritic cells were supplemented with different concentrations of reduced GSH (n = 3 per concentration) (a), or treated with glutathione reducing compound, BSO (50 μM), erastin or erastin + BSO (b) (n = 3 per condition). DMSO was used as a vehicle control. Data are representative of 4 or 6 independent experiments, c, d, Interfering with ROS may be a component but is not sufficient to reverse the enhanced efferocytosis by Slc7a11 inhibited DCs. c, WT DCs were treated with 1 μM FCCP, together with erastin, erastin or DMSO alone (n = 7 per condition) or. d, WT DCs were treated with mitoTEMPO (100 μM; n = 4) for ameliorating and scavenging ROS respectively, together with erastin (n = 4) or erastin alone (n = 4). DMSO was used as a vehicle control (n = 3). Data are representative of three or five independent experiments. All live-cell imaging data are expressed as mean ± SEM (P = 0.067, **P < 0.01; ns: non-significant; One-way ANOVA with Tukey’s multiple comparisons test).

Source Data

Extended Data Fig. 4 Analysis of dermal DCs and wound healing.

a, Gating strategy of enriched phagocytes after digestion of ears and depletion of lymphocytes. b, Immunofluorescent images of skin sections from unwounded PDGFR-GFP mice depicting Slc7a11-positive (red) cells. Nuclei were stained with DAPI. Scale bar: 50 μm. c, Annotations of innate immune cell populations arising in lesional skin. d, Frequencies of Slc7a11 expression in innate immune cells of lesional and non-lesional skin. e, Representative images of wounds of mice treated with erastin or vehicle at day 10 post-wounding. f, Wound healing dynamics comparing WT mice after full-thickness wounding and a single topical administration of apoptotic targets at the day of wounding or erastin and vehicle only at day 0 till day 2 (n = 8 per group). Data represent means ± SEM. g, Comparison of wound closure at day 2 post wounding in WT mice treated with RSL3 regimen (n = 9) versus erastin regimen (n = 7) or DMSO regimen (n = 7). Data represent means ± SEM.* P < 0.05; ** P < 0.01. One-way ANOVA with Tukey’s multiple comparisons test. Box and whiskers show minimum to maximum values with all independent replicates, center denotes median. h, i, Erastin regimen promotes in vivo migration of keratinocytes during wound healing. PDGFRa-H2BeGFP mice were treated with erastin or DMSO regimen after full-thickness wounding with an 8 mm punch biopsy. Skin sections at day 4 post-wounding were stained with h, Itga5 (n = 4 per condition, keratinocytes; n = 4 DMSO-fibroblasts; n = 7 Erastin-fibroblasts) or i, Ki67 (n = 4 per condition, keratinocytes; n = 4 DMSO-fibroblasts; n = 5 Erastin-fibroblasts) Quantification (right) and representative immunofluorescent images (left) of skin sections with respective treatments. Nuclei were stained with DAPI (blue). Violins plots show minimum to maximum values with all independent replicates, centre denotes median. (*P = 0.0420; unpaired two-tailed t-test); ns:not-significant. Scale bar: 150 μm. j, Percentage of scratch wound closure (re-epithelialization) of mouse primary Slc7a11 WT (n = 5) and KO (n = 4) keratinocytes pretreated with mitomycin C. Slc7a11KO keratinocytes show no difference indicating that the effect on migration in vivo (h) is not cell intrinsic. Data are expressed as mean ± SD.

Source Data

Extended Data Fig. 5 Gene expression patterns in Slc7a11 KO efferocytic dendritic cells.

a, Heat maps comparing efferocytic Slc7a11 KO versus WT dendritic cells showing upregulation and downregulation of differentially expressed genes (0.58 ≤ Log2FC ≤ −0.58) that are associated with metabolic and mitochondrial function, protein synthesis, ER homeostasis, transcription regulation, signaling, wound healing, cell cycle, migration and other transporters. Data are from 3-4 independent experimental replicates. Gdf15 falls under the transcriptional programs of metabolic function and regeneration, and is highlighted in red.

Source Data

Extended Data Fig. 6 Glycogen pools are altered in the absence of Slc7a11 and affect DC efferocytosis.

a, Schematic of glycogen metabolism pathway indicating the enzymes involved in glycogen breakdown, degradation and synthesis. In box, it is illustrated the inhibitory phosphorylation of Ser641, Ser645 and Ser649 on Gys1 which leads to decreased glycogen synthesis. b, Pygl siRNA targeting in dendritic cells compromises enhanced efferocytosis of Slc7a11-inhibited DCs. Live-cell imaging are expressed as mean ± SEM with n = 4 per condition, and are representative of two independent experiments. *P < 0.05; One-way ANOVA with Tukey’s multiple comparisons test. c, d, PYG inhibition via DAB compromises enhanced efferocytosis of Slc7a11-inhibited (c) or Slc7a11-KO DCs (d). Live-cell imaging are expressed as mean ± SEM with n = 3 per condition, and are representative of two independent experiments. ****P < 0.0001; One-way ANOVA with Tukey’s multiple comparisons test. e, f, Representative immunoblot (e) and quantification of glycogen metabolism enzymes (f). Data are expressed as fold change (FC) of erastin-treated BMDC to DMSO control with n = 14;Pygl and n = 13;Agl, Gys1, pGys1; **P < 0.01; ***P < 0.001, ns: non-significant via paired, two-tailed t-test.

Source Data

Extended Data Fig. 7 Metabolic inhibitors and DC efferocytosis.

a, Increased aerobic glycolysis in dendritic cells during apoptotic cell clearance and Slc7a11 inhibition. Glycolysis and OXPHOS were measured at resting dendritic cells or during efferocytosis using Seahorse XF via extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Data represent means ± SEM of n = 4 per group; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 via unpaired, two-tailed t-test or Two-way ANOVA with Tukey’s multiple comparisons test. AC: Apoptotic Cells. b, Spare capacity was measured in dendritic cells after vehicle or erastin-treatment or during efferocytosis (with Seahorse XF) using oxygen consumption rate (OCR). Data from n = 4 per group; * P < 0.05; ** P < 0.01; *** P < 0.001, via One-way ANOVA with Tukey’s multiple comparisons test. AC: Apoptotic Cells. Box and whiskers (a, b) show minimum to maximum values with all independent replicates, center denotes median. c, Assessment of cytotoxicity of drugs tested on dendritic cells by measuring Sytox Green fluorescence n = 4; DMSO, 2-DG, 3-BP; n = 2; UK5099, DON. d, Kinetics of efferocytosis by WT BMDC treated with the indicated inhibitors DON (n = 3; DMSO, Erastin, DON, DON+ Erastin) or UK5099 (n = 3; DMSO, Erastin, UK5099, UK5099+ Erastin) or CB-839 (n = 3; DMSO, CB-839; n = 6; Erastin, CB-839+ Erastin) (see schematic representation) alone or in combination with erastin. DMSO was used as a vehicle control. All live-cell imaging data are expressed as mean ± SEM and are representative of four independent experiments (*P < 0.05; ***P < 0.001; ns: not significant via One-way ANOVA with Tukey’s multiple comparisons test).

Source Data

Extended Data Fig. 8 Glycolysis/glycogen pathway inhibitors and wound healing.

a, Co-administration of CP-91149 or 3-BP with erastin regimen can reverse the accelerated wound healing in the context of Slc7a11 blockade. Wound healing dynamics of wild-type mice treated with erastin regimen or vehicle regimen consisting of a single administration to the wound site of apoptotic cells at day 0 along with erastin or DMSO vehicle given on day 0 to day 2. When indicated, the compounds CP-91149 and 3-BP were topically administered on the wounds, on day 0 to day 2. Data represent means ± SEM and show one out of two independent experiments with n = 9; DMSO regimen, n = 10; Erastin regimen, n = 8; DMSO regimen + CP-91149 or 3-BP, n = 8; Erastin regimen + CP-91149 or 3-BP (* P < 0.05; **** P < 0.0001 via Two-way ANOVA with Tukey’s multiple comparisons test).

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Extended Data Fig. 9 Targeted metabolomics profiling in Slc7a11-null or inhibited DC.

a, Peak intensity of several metabolites (absolute concentrations via targeted metabolomics) in the pellet of WT, Slc7a11 KO, and erastin-treated dendritic cells at steady state or upon efferocytosis (n = 4 biological replicates per condition; ****P < 0.0001 via One-way ANOVA with Tukey’s multiple comparisons test). Box and whiskers show minimum to maximum values with all independent replicates, center denotes median.

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Extended Data Fig. 10 Erastin ameliorates corpse clearance in the wounds of db/db mice and promotes partial wound healing in GDF15 KO mice.

a, b, Erastin ameliorates corpse clearance with or without the addition of apoptotic bolus in in the wounds of db/db mice. Quantification of apoptotic cleaved caspase-3+ cells in wounded skin of a, normoglycemic (B6) mice at day 4 post-wounding and b, diabetic (db/db) mice at day 8 post-wounding treated with erastin or vehicle regimen versus erastin or DMSO (for vehicle control) without the single administration of the apoptotic bolus. (B6: n = 4; UNW; n = 10; Vehicle regimen; n = 11; Erastin regimen; n = 4; Vehicle or Erastin; db/db: n = 5 mice per group; * P < 0.05; ** P < 0.01 with unpaired, two-tailed t-test). c, Lysates were prepared using bone-marrow derived dendritic cells from WT, GDF15 KO and Slc7a11 KO mice, mRNA was isolated, followed by RT-qPCR analysis for Slc7a11. (n = 5 per genotype; data are presented as fold change to Slc7a11 expression to control (WT) BMDC). d, GDF15 deficiency does not impair DC efferocytosis. Kinetics of efferocytosis by GDF15 KO and WT BMDC. Live-cell imaging data are expressed as mean ± SEM with n = 12; WT, n = 8; GDF15KO. e, Erastin regimen promotes partial wound healing in GDF15 KO mice. Wound healing dynamics comparing GDF15 KO and littermate control mice treated with erastin or DMSO vehicle given on day 0 to day 2. All wound sizes are expressed as percentage of initial wound size at day 2 post-wounding. (n = 8; WT+Erastin, n = 9; WT+vehicle, KO+Vehicle, KO+Erastin; * P < 0.05; ** P < 0.01; between groups, via unpaired, two-tailed t-test. Violin plots (c), and the box and whiskers plots (a, b, e) show the minimum to maximum values with all independent replicates, center denotes median.

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Extended Data Table 1 Top 10 predictions for Slc7a11 associated diseases via ARCHS4

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Maschalidi, S., Mehrotra, P., Keçeli, B.N. et al. Targeting SLC7A11 improves efferocytosis by dendritic cells and wound healing in diabetes. Nature 606, 776–784 (2022). https://doi.org/10.1038/s41586-022-04754-6

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