Polyamine import and accumulation causes immunomodulation in macrophages engulfing apoptotic cells

Abstract

Phagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue homeostasis and drives anti-inflammatory programming in engulfing macrophages. Here, we assess metabolites in naive and inflammatory macrophages following engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to increases in the arginine-derived polyamines, spermidine and spermine, in vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does not arise from retention of apoptotic cell metabolites or de novo synthesis but from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase. Blocking polyamine import prevents efferocytosis from suppressing macrophage interleukin (IL)-1β or IL-6. This identifies efferocytosis as a trigger for polyamine import and accumulation, and imported polyamines as mediators of efferocytosis-induced immune reprogramming.

Keywords: apoptosis; arginine; efferocytosis; macrophage; metabolites; phagocytosis; polyamines.

Figure 1.. UHPLC-MS identifies intracellular metabolites in macrophages after engulfment of apoptotic, live, or necrotic target cells

(A) Timeline for co-culture and sorting. (B) LPS-primed murine peritoneal macrophages were cultured alone (no target) or co-cultured with ApoJ, (IgGJ), or NecJ Jurkat cells, then FACS sorted into non-engulfing and engulfing populations. Macrophage metabolites were assessed using UHPLC-MS. Heatmaps show intracellular metabolite levels after target-cell engulfment relative to the no-target group (red, enriched; blue, reduced). Two time points were assessed (4 h, pink; 8 h, green) with n = 3 replicates at each time point. Scale bars in microscopy images represent 25 μm.

Figure 2.. Polyamines accumulate in macrophages after efferocytosis but not in response to engulfment of other target cells in vitro and in vivo

(A) Polyamine synthesis pathway from arginine. (B and C) Metabolites in (B) non-engulfing and (C) engulfing LPS-primed murine peritoneal macrophages after co-culture with target cells. n = 5. (D) Metabolites in LPS-primed peritoneal macrophages co-cultured with PS-containing liposomes or carboxylated latex beads. Data show fold change of PS-liposome exposed macrophages or carboxylated bead engulfing unexposed macrophages (ApoJ target response included for comparison). n = 3–6. (E) ApoJ were injected intraperitoneally (IP) into naive mice. Engulfing peritoneal macrophages were collected 8 h later; metabolites were compared with control macrophages from un-injected mice. n = 3. (F) IgGJ were injected IP into naive mice. Engulfing peritoneal macrophages were collected 8 h later; metabolites were compared with control macrophages from un-injected mice. n = 3. (G and H) Bone marrow chimeras were generated to track endogenous apoptotic cell engulfment. (G) Mixed bone marrow chimera scheme to generate mice with representative FACS showing cytospin images of sorted non-engulfing and engulfing peritoneal macrophages. Scale bars in microscopy images represent 10 μm. (H) Mixed bone marrow chimeras were treated with IP thioglycolate and lavage harvested after 48 h. Metabolites from CD45.1⁺ CD45.2⁻DsRed⁺ engulfing macrophages were compared with metabolites from CD45.1⁺CD45.2⁻DsRed⁻ non-engulfing macrophages. n = 4. Data are shown as mean ± standard error of the mean (SEM); *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.. Target-cell-derived amino acids but not polyamines accumulate in macrophages after engulfment, and arginine metabolism does not explain polyamine accumulation

(A) Timeline for Iso-ApoJ co-culture; ApoJ targets were loaded with [¹³C,¹⁵N] amino acids then co-cultured with macrophages. (B and C) [¹³C,¹⁵N] label was traced in macrophages to identify ApoJ-derived metabolites in amino acids (B) and polyamines (C). (D) Timeline to trace arginine flux; following co-culture with ApoJ targets, macrophage medium was switched to medium containing [¹³C₆,¹⁵N₄] arginine. (E–G) Relative flux was calculated by comparing the fraction of [¹³C,¹⁵N] isotopologue in a downstream metabolite with the fraction in its upstream precursor. Dashed line shows baseline of control no-target macrophages. (E) Relative ratio to indicate arginase activity. (F) Relative ratio to indicate ODC1 activity. (G) Relative ratio to indicate spermidine synthase activity. n = 4/group. LPS-primed peritoneal macrophages were used for all experiments. Data are shown as mean ± SEM; **p < 0.01, ***p < 0.001.

Figure 4.. Efferocytosis increases polyamine import via a Rac1 and PI3 kinase-dependent mechanism, leading to polyamine accumulation and IL-1β suppression

(A) Timeline to track polyamine import; following co-culture with targets, macrophage medium was switched to medium containing fluorescent spermine (NBD-spermine). (B and C) Polyamine import in response to ApoJ (red), IgGJ (blue), and beads (purple). (B) Representative histogram plots following target co-cultures: engulfing (dark color), non-engulfing (light color), and no-target control (dark grey). Macrophages without NBD-spermine shown in light grey. (C) Mean fluorescence intensity (MFI) of NBD-spermine quantified for engulfing macrophages after 7 h. n = 4–6. (D) Effect of AMXT-1483 pre-treatment on NBD-spermine import; MFI of NBD-spermine quantified for control and ApoJ-engulfing macrophages after 7 h. n = 4–5. (E) Effect of inhibitor pre-treatment on import of NBD-spermine; MFI of NBD-spermine quantified after 7 h. n = 3–12. (F and G) Spermine import in vivo. Naive mice were given IP injections of ApoJ, followed by NSC23766 1 h later at 5 mg/kg, followed by NBD-spermine. (F) Representative histogram of NBD-spermine import by engulfing (dark red), engulfing + NSC23766 (light blue), and no-target control macrophages (dark grey). Macrophages without NBD-spermine are shown in light grey. (G) MFI of NBD-spermine quantified after 5 h. (H) Import of NBD-spermine compared with import of AF647-dextran after efferocytosis. n = 8–12. (I) Macrophages were cultured ± ApoJ for 1 h, ± NSC23766 medium for 7 h, then sorted by FACS into non-engulfing and engulfing populations. Macrophage metabolites were assessed using UHPLC-MS shown as fold change relative to the no-target group. n = 3–6. (J and K) BMDMs were cultured alone or co-cultured with ApoJ. After removal of ApoJ, BMDMs were treated with control media, LPS, or LPS + NSC23766. RNA was collected 6 h later and (J) il-1b or (K) il-6 expression was assessed by RT-PCR, normalized to b2m expression. n = 4–5. Data are shown as mean ± SEM; **p < 0.01, ***p < 0.001.