Abstract
The clearance of apoptotic cells by macrophages (efferocytosis) prevents necrosis and inflammation and activates pro-resolving pathways, including continual efferocytosis. A key resolution process in vivo is efferocytosis-induced macrophage proliferation (EIMP), in which apoptotic cell-derived nucleotides trigger Myc-mediated proliferation of pro-resolving macrophages. Here we show that EIMP requires a second input that is integrated with cellular metabolism, notably efferocytosis-induced lactate production. Lactate signalling via GPR132 promotes Myc protein stabilization and subsequent macrophage proliferation. This mechanism is validated in vivo using a mouse model of dexamethasone-induced thymocyte apoptosis, which elevates apoptotic cell burden and requires efferocytosis to prevent inflammation and necrosis. Thus, EIMP, a key process in tissue resolution, requires inputs from two independent processes: a signalling pathway induced by apoptotic cell-derived nucleotides and a cellular metabolism pathway involving lactate production. These findings illustrate how seemingly distinct pathways in efferocytosing macrophages are integrated to carry out a key process in tissue resolution.
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Data availability
All data supporting the present study are available within the paper and supplementary information files. Source data are provided with this paper.
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Acknowledgements
This work was supported by an American Heart Association Postdoctoral Fellowship (grant no. 900337 to M.S.); the Niels Stensen Fellowship (to M.S.) and NIH/NHLBI grant nos. R35-HL145228 and P01-HL087123 (to I.T.). We thank L. Becker (University of Chicago) for providing Ldhafl/fl and Ldhafl/fl; LysMCre+/− mouse femurs for BMDM differentiation to use in our in vitro studies. We thank X. Wang (Columbia University) for assisting with intravenous injections for BMT. We acknowledge C. Lu of the Columbia Center for Translational Immunology Core Facility for assisting in the immunofluorescent imaging experiments that were conducted in the Columbia Center for Translational Immunology Core Facility, funded by NIH grant nos. P30CA013696, S10RR027050 and S10OD020056.
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D.N. and I.T. conceived the project. D.N., M.S. and I.T. provided intellectual input to the development of the project. D.N. performed the in vitro experiments. D.N. and M.S. performed the in vivo dexamethasone-thymus experiment.
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Extended data
Extended Data Fig. 1 Related to Figs. 1 and 2: Controls for BMDM experiments.
a, BMDMs were incubated with or without ACs for 45 minutes before washing and chasing for 1 hour in low-serum DMEM ± 50 µM FX11. The media were assayed for lactate concentration (n = 3). b, BMDMs were transfected with scrambled (Scr) or Ldha siRNA for 72 hours and then assayed for Ldha mRNA by RT-qPCR (n = 3). c) BMDMs pre-treated with 50 µM FX11 for 1 hour were incubated for 45 minutes with PKH26-labelled ACs and quantified for % PKH26+ macrophages (n = 3). d, BMDMs were incubated with ACs for 45 minutes, chased for 3 hours ± 50 µM FX11 and ± 2/5/10 mM LA, and immunoblotted for Myc (n = 3). e, BMDMs were incubated with or without ACs for 45 minutes, chased for 3 hours ± 50 µM FX11 and ± 10 mM sodium lactate (NaLa), and immunoblotted for Myc (n = 3). f, BMDMs transfected with 50 nM scrambled or Myc siRNA for 72 hours were incubated with or without ACs for 45 minutes, chased for 3 hours, and immunoblotted for Myc (n = 3). g, BMDMs were treated ± 10 mM LA for 3 hours and immunoblotted for Myc (n = 3). h, BMDMs were treated for 24 hours ± 10 mM LA before performing a cell count (n = 3). i, BMDMs were incubated with or without ACs for 45 minutes, chased for 3 hours ± 10 mM LA, and immunoblotted for Myc (n = 3). j, BMDMs were incubated with ACs for 45 minutes, chased for 3 hours ± 50 µM FX11 and ± 10 or 25 mM LA, and immunoblotted for Myc. Similar results were obtained in a repeat experiment. k, BMDMs were incubated with or without ACs for 45 minutes, chased for 3 hours ± 10 µM MG132, and immunoblotted for Myc (n = 3). Bars represent means ± s.e.m. Statistics were performed by two-tailed student’s t-test in panels b-c, h, or one-way ANOVA in panels a, d, e, i, and k. n.s. = non-significant (P > 0.05).
Extended Data Fig. 2 Related to Figs. 3 and 4: Controls for BMDM experiments.
a, BMDMs were transfected with 50 nM scrambled or Sirt1 siRNA for 72 hours, incubated with PKH26-labelled ACs for 45 minutes, and quantified for the percent PKH26+ macrophages (n = 3). b, BMDMs were incubated with or without ACs for 45 minutes, chased for 3 hours ± 10 µM EX527 and ± 10 mM LA, and immunoblotted for Myc (n = 3). c, BMDMs were treated with 50 ng/mL CSF-1 for 3, 6, 12, or 24 hours and immunoblotted for Myc. The image presented is one representative replicate (n = 3). d, BMDMs were treated with or without 50 ng/mL CSF-1 + 50 µM FX11 or 10 µM EX527, and immunoblotted for Myc (n = 3). e, BMDMs were treated for 24 hours with 50 ng/mL CSF-1 + 50 μM FX11 or 10 μM EX527, and quantified for cell number (n = 3). f, BMDMs were transfected with 50 nM scrambled or Myc siRNA for 72 hours, incubated ± ACs for 45 minutes or with CSF-1, and then assayed 24 hours later for cell number and Myc protein for CSF1-treated cells (n = 3). g, BMDMs pre-treated for 1 hour with 10 µM CompC were incubated with PKH26-labelled ACs and quantified for the percent PKH26+ (n = 3). h, BMDMs were chased for 1 hour + 10 µM CompC with or without 500 µM NMN and then assayed for NAD+ to NADH ratio (n = 3). i, BMDMs were treated ± 500 µM NMN for 3 hours and then immunoblotted for Myc (n = 3). j, BMDMs transfected with scrambled or Sirt1 siRNA were chased for 3 hours ± 500 µM NMN and then immunoblotted for Myc (n = 3). Bars represent means ± s.e.m. Statistics were performed by two-tailed student’s t-test in panels a and g, or one-way ANOVA in panels b-f, h, and j. n.s. = non-significant (P > 0.05).
Extended Data Fig. 3 Related to Fig. 5: Controls for BMDM experiments.
a, BMDMs were transfected with 50 nM scrambled or Slc16a1 siRNA for 72 hours and then assayed for Slc16a1 mRNA by RT-qPCR (n = 3). b, BMDMs transfected with 50 nM scrambled or Slc16a1 siRNA for 72 hours were incubated with ACs for 45 minutes, chased for 3 hours in 1% FBS DMEM before collecting supernatants and cell lysates to measure intracellular and extracellular lactate concentrations (n = 3). c, BMDMs were transfected with 50 nM scrambled or Gpr132 siRNA for 72 hours and then assayed for Gpr132 mRNA by RT-qPCR (n = 3). d, BMDMs transfected with 50 nM scrambled or Gpr132 siRNA for 72 hours were incubated with or without ACs for 45 minutes, chased for 3 hours ± 50 µM FX11 and ± 10 mM LA, and immunoblotted for Myc. The displayed immunoblot is a representative replicate (n = 3). e, BMDMs were incubated with or without ACs for 45 minutes, chased for 3 hours ± 50 μM FX11, and assayed for Gpr132 mRNA (n = 3). Bars represent means ± s.e.m. Statistics were performed by two-tailed student’s t-test in panels a-c, one-way ANOVA in panel d, or two-way ANOVA in panel e. n.s. = non-significant (P > 0.05).
Extended Data Fig. 4 Related to Fig. 6: Controls and blood counts for the dexamethasone-thymus experiment.
a-f, Counts of blood WBCs, neutrophils, lymphocytes, monocytes, eosinophils, and basophils (n = 5). g, Plasma was measured for lactate concentration (n = 5). h, Mac2+ cells in immunostained thymus sections were counted per field of view (FOV) using images taken with a 20x objective (n = 8,6). Bars represent means ± s.e.m. Statistics were performed by two-tailed student’s t-test in panels a-e and g-h, and Mann–Whitney test for panel f. n.s. = non-significant (P > 0.05).
Supplementary information
Supplementary Table 1
qPCR primer sequences.
Source data
Source Data Fig. 1–6 and Extended Data Figs. 1–3
Unprocessed western blots for Figs. 1–6 and Extended Data Figs. 1–3.
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Ngai, D., Schilperoort, M. & Tabas, I. Efferocytosis-induced lactate enables the proliferation of pro-resolving macrophages to mediate tissue repair. Nat Metab 5, 2206–2219 (2023). https://doi.org/10.1038/s42255-023-00921-9
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DOI: https://doi.org/10.1038/s42255-023-00921-9