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Efferocytosis drives a tryptophan metabolism pathway in macrophages to promote tissue resolution

Nature Metabolism volume 6, pages 1736–1755 (2024)Cite this article

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A Publisher Correction to this article was published on 16 September 2024

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Abstract

Macrophage efferocytosis prevents apoptotic cell (AC) accumulation and triggers inflammation-resolution pathways. The mechanisms linking efferocytosis to resolution often involve changes in macrophage metabolism, but many gaps remain in our understanding of these processes. We now report that efferocytosis triggers an indoleamine 2,3-dioxygenase-1 (IDO1)-dependent tryptophan (Trp) metabolism pathway that promotes several key resolution processes, including the induction of pro-resolving proteins, such interleukin-10, and further enhancement of efferocytosis. The process begins with upregulation of Trp transport and metabolism, and it involves subsequent activation of the aryl hydrocarbon receptor (AhR) by the Trp metabolite kynurenine (Kyn). Through these mechanisms, macrophage IDO1 and AhR contribute to a proper resolution response in several different mouse models of efferocytosis-dependent tissue repair, notably during atherosclerosis regression induced by plasma low-density lipoprotein (LDL) lowering. These findings reveal an integrated metabolism programme in macrophages that links efferocytosis to resolution, with possible therapeutic implications for non-resolving chronic inflammatory diseases, notably atherosclerosis.

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Fig. 1: Macrophage efferocytosis drives Trp metabolism and IDO1 expression, which are dependent on AC degradation and the amino acid transporter SLC36A4.
Fig. 2: IDO1 deficiency or inhibition suppresses continuing efferocytosis and post-efferocytic resolving mediator induction.
Fig. 3: Continuing efferocytosis and resolving mediator induction require SLC36A4 and the Trp metabolite Kyn.
Fig. 4: Role of IDO1 and Kyn in resolution in vivo.
Fig. 5: AC-derived Kyn induces Tgfb1 and Il10 expression and promotes continuing efferocytosis through an AhR pathway.
Fig. 6: Kyn requires ERK1/2 for AhR activation and resolution signalling in efferocytic macrophages.
Fig. 7: Macrophage AhR promotes efferocytosis and resolution in vivo.
Fig. 8: Macrophage IDO1 contributes to efferocytosis and features of plaque stabilization in atherosclerosis regression.

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All data supporting the study are available in the manuscript and supplementary information. Source data are provided with this paper.

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Acknowledgements

We thank R. Ramakrishnan (Columbia University) for his guidance on statistical analysis of the data in this study. We thank B. Gerlach for his invaluable initial discussions, which greatly contributed to the development of this research. This work was supported by NIH/NHLBI grant nos. R35-HL145228 and P01-HL087123 (to I.T.) and R01-HL159012 (to J.S. and I.T.). D.N. was supported by American Heart Association postdoctoral award no. 24POST1192241. Immunofluorescence imaging experiments were conducted in the Columbia Center for Translational Immunology Core Facility, funded by NIH grant nos. P30CA013696, S10RR027050 and S10OD020056. Flow cytometry experiments were conducted using the Herbert Irving Comprehensive Cancer Center Flow Cytometry Shared Resources, funded in part through NIH grant no. P30CA013696. Samples for histological analysis were prepared in the Molecular Pathology Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grant no. P30CA013696. The confocal microscopy work in this study was conducted in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grants nos. P30CA013696 and S10RR025686. This work was supported in part by the Proteomics & Metabolomics Core at Moffitt Cancer Center and funded as part of an NCI-designated Comprehensive Cancer Center (P30 CA076292).

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Authors and Affiliations

  1. Department of Medicine, Columbia University Irving Medical Center, New York, NY, USA

    Santosh R. Sukka, Patrick B. Ampomah, David Ngai, Xiaobo Wang, George Kuriakose & Ira Tabas

  2. Proteomics and Metabolomics Core, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA

    Lancia N. F. Darville & John M. Koomen

  3. Center for Nanomedicine and Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Yuling Xiao & Jinjun Shi

  4. Department of Animal Sciences, Integrative Immunology and Behavior Program and Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Robert H. McCusker

  5. Departments of Physiology and Cell Biology, Columbia University Irving Medical Center, New York, NY, USA

    Ira Tabas

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  1. Santosh R. Sukka
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Contributions

S.R.S. and I.T. conceived the project. P.B.A., D.N., X.W., J.S. and R.H.M. provided additional intellectual input in the development of the project. L.N.F.D. conducted the LC–MS analyses under the guidance of J.M.K. G.K. and Y.X. helped with the mouse atherosclerosis experiments. S.R.S. and I.T. wrote the manuscript and the other co-authors provided comments and revisions.

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Correspondence to Santosh R. Sukka or Ira Tabas.

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Extended data

Extended Data Fig. 1 Related to Fig. 1: Macrophage efferocytosis drives Trp metabolism.

a, Tryptophan and kynurenine content expressed as pmol/μg cell protein in BMDMs incubated ± ACs from the experiment in Fig. 1a (n = 6 biological replicates/group). b,c, Additional metabolite values from BMDMs incubated ± ACs from the experiment in Fig. 1a (n = 6 biological replicates/group). Data are mean ± SEM, and significance was determined by two-tailed Student’s t-test.

Extended Data Fig. 2 Related to Fig. 1: Examples of extracted ion chromatograms.

a, Reversed phase separation and mass spectrometry detection of 12 neat tryptophan metabolite standards (10 ng) individually analyzed. Each trace shows ion signal at a given mass-to-charge ratio (m/z) as the compounds elute from the Atlantis T3 reversed phase column. b, Reversed phase separation on an Atlantis T3 reversed phase column and mass spectrometry detection of tryptophan metabolites from an AC- wild type sample. Each trace shows ion signal at a given mass-to-charge ratio (m/z) with a mass tolerance of 5 ppm. For both sets of chromatograms, data are shown for: (i) 2−picolinic acid), (ii) nicotinic acid, (iii) nicotinamide, (iv) quinolinic acid, (v) nicotinamide adenine dinucleotide (NAD)+, (vi) serotonin, (vii) DL-kynurenine, (viii) N-formylkynurenine, (ix) L-tryptophan, (x) tryptamine, (xi) kynurenic acid, and (xii) anthranilic acid. The normalization level (NL) indicates the intensity of the base peak for each spectrum. In b, although more background was observed in the extracted ion chromatogram for anthranilic acid, the anthranilic acid signal is easily differentiated from the background.

Extended Data Fig. 3 Related to Fig. 1: Absolute quantitation of kynurenine and tryptophan.

a-d, Quantitation using a known amount of isotope-labeled standards. a,b, Examples of extracted ion chromatograms showing reversed phase separation and mass spectrometry detection for kynurenine (10 ng) and D4-kynurenine (50 ng); and tryptophan (10 ng) and 13C11-tryptophan (50 ng). Each trace shows the ion signal at a given mass-to-charge ratio (m/z) as the compounds elute from an Atlantis T3 reversed phase column. c,d, Positive ion mass spectra of kynurenine and D4-kynurenine; and tryptophan and 13C11-tryptophan. e-h, Quantitation of kynurenine and tryptophan in an AC− wild type sample. e,f, Examples of extracted ion chromatograms showing reversed phase separation and mass spectrometry detection for endogenous kynurenine and spiked D4-kynurenine (50 ng); and endogenous tryptophan and spiked 13C11-tryptophan (50 ng). Each trace shows the ion signal at a given mass-to-charge ratio (m/z) as the compounds elute from an Atlantis T3 reversed phase column. g,h, Positive ion mass spectra of endogenous kynurenine and D4-kynurenine; and endogenous tryptophan and 13C11-tryptophan. The normalization level (NL) indicates the intensity of the base peak for each spectrum.

Extended Data Fig. 4 Related to Fig. 1: The role of SLC36A4 in Trp metabolism in efferocytosing macrophages.

a, Immunofluorescence microscopy of SLC36A4 (green) and LAMP-1 (red) in Scr- or siSlc36a4-transfected BMDMs incubated 45 mins with PKH26-labelled ACs (pseudocolored white); DAPI (blue) nuclear stain. Scale bar, 50 μm. Another set of cells was assayed for Slc36a4 mRNA and immunoblotted for SLC36A4 (n = 3 biological replicates/group). b, Immunofluorescence microscopy of SLC36A4 (green) in BMDMs incubated with PKH26-labelled ACs (red) for 45 min. Scale bar, 50 μm. White arrows, engulfed ACs; blue arrows, unengulfed ACs. c, Representative image of Scr-transfected macrophages not incubated with ACs and then stained for SLC36A4 (green) and DAPI (blue); note low expression of SLC36A4 compared with AC+ macrophages in Fig. 1b. Image is representative of 3 biological triplicates. Scale bar, 50 μm. d, BMDMs were incubated ± apoptotic Jurkat cells (apJCs) or apoptotic macrophages (apMϕs), chased for 3 h, and assayed for Slc36a4 (n = 6 biological replicates/group). e, BMDMs were incubated ± ACs or PS-beads for 1 h, chased for 3 h, and immunoblotted for SLC36A4. f, BMDMs pre-treated with ± 20 µM MG132 were incubated ± ACs for 1 h, chased for 3 h, and immunoblotted for SLC36A4. g, Scr- or siSlc36a4-transfected BMDMs were incubated with PKH26-labelled ACs (red) and quantified for the percentage of PKH26+ macrophages (arrows) of total macrophages. Scale bar, 50 μm (n = 5 biological replicates/group). h, Tryptophan and kynurenine in Scr- or siSlc36a4-transfected BMDMs incubated with ACs (see Fig. 1d; n = 6 biological replicates/group). i, BMDMs were incubated ± apoptotic macrophages for 1 h and assayed for Ido1 (n = 3 biological replicates/group). j, Scr- or siSLC36A4-transfected HMDMs were for SLC36A4 (n = 3 biological replicates/group). All mRNA data are expressed relative to the indicated control groups. Data are mean ± SEM, and significance was determined by two-tailed Student’s t-test or one-way ANOVA with Fisher’s LSD post-hoc analysis.

Source data

Extended Data Fig. 5 Related to Figs. 2 and 3: Additional data on the roles of SLC36A4 and IDO1 in resolution.

a, Control or IDO1-KO BMDMs incubated with PKH26-labelled ACs were quantified for percent PKH26+ macrophages (arrows). Scale bar, 50 μm (n = 6 biological replicates/group). b, Scr- or siIdo1-transfected BMDMs incubated with pHrodo-Red-labelled ACs were quantified for percent pHrodo-Red+ macrophages of total macrophages by flow cytometry (n = 3 biological replicates/group); immunoblotted for IDO1 (n = 3 samples/group); and assayed for Ido1 (n = 6 biological replicates/group). c, Flow cytometry contour plots for the experiment in Fig. 2b. d, BMDMs treated with ACs ± epacadostat were assayed for Arg1 and Mcf2 after a 6-h or 2-h chase, respectively (n = 3 biological replicates/group). e,f, Contour plots for the experiments in Fig. 2g, h. g, Scr- or siIDO1-transfected HMDMs were assayed for IDO1 (n = 3 biological replicates/group). h, Scr- or siSlc36a4-transfected BMDMs were incubated ± apoptotic macrophages, chased for 6 h, and assayed for Tgfb1 and Il10 (n = 3 biological replicates/group). i, Scr- or siSlc36a4-transfected BMDMs were pre-treated ± kynurenine and then incubated ± ACs for 1 h, chased for 6 h in Trp-deficient medium, and assayed for Tgfb1 and Il10 mRNA. Right, normal and Trp-depleted media were assayed for Trp by LC−MS/MS (right) (n = 3 biological replicates/group). j, BMDMs pre-treated for 1 h with vehicle or 50 µM Trp were incubated ± ACs, chased for 6 h, and assayed for Tgfb1 and Il10 (n = 3 biological replicates/group). k, Scr- or siSlc36a4-transfected BMDMs were pre-treated ± 50 μM Trp for 1 h, incubated ± ACs. and assayed for Tgfb1 and Il10 (n = 3 biological replicates/group). All mRNA data are expressed relative to the indicated control groups. Data are mean ± SEM, and significance was determined by two-tailed Student’s t-test or one-way ANOVA with Fisher’s LSD post-hoc analysis.

Source data

Extended Data Fig. 6 Related to Figs. 4 and 5: Additional in-vivo and in-vitro data on the IDO1-Kyn-AhR pathway.

a-c, The thymi of the mice from Fig. 4g–e were immunostained for IDO1 (red) and Mac2 (green) and quantified for IDO1 MFI in Mac2+ areas (arrows). Also shown are thymus weight, thymus cellularity, and F4/80+ macrophages/thymus (n = 8 mice/group). d, Control and Ido1−/− BMDMs (top 2 graphs), or Scr- and siSlc364-transfected BMDMs (bottom graph), were pre-treated for 1 h ± Kyn and incubated ± ACs. After a 6-h chase, the cells were assayed for Cyp1a1 and Cyp1b1 (n = 3 biological replicates/group). e, BMDMs were incubated ± apoptotic macrophages and assayed for Cyp1a1. f, Scr- or siSLC36A4-transfected HMDMs were incubated ± ACs, chased for 3 h, and assayed for CYP1A1 and CYP1B1 (n = 3 biological replicates/group). g, Scr- or siAhr-transfected BMDMs were incubated with DiD-labelled ACs and quantified for percent DiD+ macrophages (arrows). Scale bar, 50 μm (n = 4 biological replicates/group). h, BMDMs pre-treated ± CH223191 were assayed for continuing efferocytosis as in Fig. 2a. Arrows, PKH26+PKH67+ macrophages. Scale bar, 50 μm (n = 3 biological replicates/group). i, As in panel h, but one of the cohorts was also treated with cytochalasin D before the second round of efferocytosis. Arrows, PKH26+PKH67+ macrophages. Scale bar, 50 μm (n = 3 biological replicates/group). j, Scr- or siAhr-transfected BMDMs were assayed for Ahr (n = 3 biological replicates/group) and immunoblotted for AhR protein (n = 3 samples/group). k, Scr- or siAHR-transfected HMDMs were assayed for AHR (n = 3 biological replicates/group). l, Scr- or siArnt-transfected BMDMs were assayed for Arnt (n = 3 biological replicates/group). All mRNA data are expressed relative to the indicated control groups. Data are mean ± SEM, and significance was determined by two-tailed Student’s t-test or one-way ANOVA with Fisher’s LSD post-hoc analysis.

Source data

Extended Data Fig. 7 Related to Figs. 6 and 7: Additional in-vivo and in-vitro data on the role of AhR in efferocytosis-induced resolution.

a, BMDMs were incubated for 1 h with 100 μM Kyn alone or with control (non-PS) or PS beads ± Kyn and then assayed for Cyp1a1 and Ido1 after a 3-h chase (n = 4 samples/group). b, BMDMs incubated with ACs, PS-beads (PS), Kyn, or PS-beads + Kyn were assayed for Tgfb1 after a 6-h chase (n = 4 samples/group). c, BMDMs incubated with 50 μM Trp, PS-beads (PS), Trp and PS-beads, or 100 μM Kyn and PS-beads for 1 h were assayed for Cyp1a1 and Il10 after a 3-h or 6-h chase, respectively (n = 3 biological replicates/group). d, BMDMs pre-treated ± U0126 were incubated with ACs for 1 h and then immunoblotted for AhR and ARNT after a 3-h chase (n = 3 samples/group). e, BMDMs treated with PS-beads (PS) and Kyn ± U0126 for 1 h were assayed for Tgfb1 or Ido1 after a 3-h chase or 6-h chase, respectively (n = 3 biological replicates/group). f, BMDMs incubated ± ACs ± CH223191 for 1 h were assayed Hsp90 or Xap2 after a 3-h chase (n = 3 biological replicates/group). g, Scr-, siHsp90-, or siXap2-transfected BMDMs were incubated with PKH26-labelled ACs and quantified for percent PKH26+ macrophages (arrows). Scale bar, 50 μm (n = 3 biological replicates/group). h, Proposed pathway (created using BioRender.com): Trp form an efferocytosed AC (AC1) is transported into the macrophage by SLC36A4 and then converted to Kyn by IDO1. Kyn and activated ERK induce Hsp90 and Xap2, leading to AhR-ARNT-mediated transcription of Tgfb1, Il10, and Ido1 and Rac1-mediated AC2 internalization (continuing efferocytosis). i-k, The thymi of the mice from Fig. 7 were immunostained for AhR (red) and Mac2 (green) and quantified for AhR MFI in Mac2+ areas (arrows). Also shown are thymus weight and F4/80+ macrophages/thymus (n = 8 mice/group). All mRNA data are expressed relative to the indicated control groups. Data are mean ± SEM, and significance was determined by two-tailed Student’s t-test or one-way ANOVA with Fisher’s LSD post-hoc analysis.

Source data

Extended Data Fig. 8 Related to Fig. 8: Systemic and lesional parameters in control and Mϕ-IDO1-iKO BMT Ldlr−/− mice.

Ldlr−/− mice were transplanted with BM from Ido1fl/fl (Control) or Ido1fl/flCx3cr1creERT2+/− (Mϕ-IDO1-iKO) mice and then fed the Western diet for 16 weeks. One cohort from each group was harvested (Baseline), and the rest of the mice were switched to chow diet, injected with HDAd-LDLR virus, and given tamoxifen. After 7 weeks, the mice were harvested (Regression). a-k, Body weight, total plasma cholesterol, fasting blood glucose, complete blood count (n = 9–10 mice/group). WBC, white blood cell; NE, neutrophils; LY, lymphocytes; MO, monocytes; EO, eosinophils; BA, basophils, RBC, red blood cells; PLT, platelets. l, Immunostaining of IDO1 (red) and Mac2 (green) in regressing aortic root lesions, with quantification of IDO1 MFI in Mac2+ and Mac2− areas. Arrows, examples of IDO1-Mac2 co-localization. DAPI was used for nuclear staining. Scale bar, 25 μm (n = 10 mice/group). m, Quantification of lesion area, based on H&E staining of the aortic root lesions (n = 10 mice/group). n, The regressing aortic root lesions of Control and Mϕ-IDO1-iKO groups were immunostained for Mac2 (macrophages; green) and TGF-β1 or IL-10. (red) Arrows, examples of colocalization of Mac2 and TGF-β1 (top) and Mac2 and IL-10 (bottom). DAPI (blue) was used for nuclear staining. Scale bar, 50 μm. o, The total number of Mac2+ cells per lesion section was quantified in regressing aortic root lesions (n = 10 mice/group). The data are expressed as mean ± SEM, and significance was determined by one-way ANOVA with Fisher’s LSD post-hoc analysis for panels a-k and m, and by Student’s t-test for panels l and o.

Supplementary information

Supplementary Tables 1–5

Supplementary Table 1: Macrophage efferocytosis drives tryptophan metabolism. Supplementary Table 2: The average and s.d. of retention time and m/z of tryptophan metabolites using LC–MS. Supplementary Table 3: Macrophage efferocytosis drives tryptophan metabolism in an SLC36A4-dependent manner. Supplementary Table 4: List of antibodies. Supplementary Table 5: Primer sequences and RNAi list.

Reporting Summary

Source data

Source Data Figs. 1, 5, and 6

Unprocessed western blots for Figs. 1, 5, and 6.

Source Data Extended Data Fig. 4–7

Unprocessed western blots for Extended Data Figs. 4–7.

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Sukka, S.R., Ampomah, P.B., Darville, L.N.F. et al. Efferocytosis drives a tryptophan metabolism pathway in macrophages to promote tissue resolution. Nat Metab 6, 1736–1755 (2024). https://doi.org/10.1038/s42255-024-01115-7

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