Efferocytosis potentiates the expression of arachidonate 15-lipoxygenase (ALOX15) in alternatively activated human macrophages through LXR activation

Abstract

Macrophages acquire anti-inflammatory and proresolving functions to facilitate resolution of inflammation and promote tissue repair. While alternatively activated macrophages (AAMs), also referred to as M2 macrophages, polarized by type 2 (Th2) cytokines IL-4 or IL-13 contribute to the suppression of inflammatory responses and play a pivotal role in wound healing, contemporaneous exposure to apoptotic cells (ACs) potentiates the expression of anti-inflammatory and tissue repair genes. Given that liver X receptors (LXRs), which coordinate sterol metabolism and immune cell function, play an essential role in the clearance of ACs, we investigated whether LXR activation following engulfment of ACs selectively potentiates the expression of Th2 cytokine-dependent genes in primary human AAMs. We show that AC uptake simultaneously upregulates LXR-dependent, but suppresses SREBP-2-dependent gene expression in macrophages, which are both prevented by inhibiting Niemann-Pick C1 (NPC1)-mediated sterol transport from lysosomes. Concurrently, macrophages accumulate sterol biosynthetic intermediates desmosterol, lathosterol, lanosterol, and dihydrolanosterol but not cholesterol-derived oxysterols. Using global transcriptome analysis, we identify anti-inflammatory and proresolving genes including interleukin-1 receptor antagonist (IL1RN) and arachidonate 15-lipoxygenase (ALOX15) whose expression are selectively potentiated in macrophages upon concomitant exposure to ACs or LXR agonist T0901317 (T09) and Th2 cytokines. We show priming macrophages via LXR activation enhances the cellular capacity to synthesize inflammation-suppressing specialized proresolving mediator (SPM) precursors 15-HETE and 17-HDHA as well as resolvin D5. Silencing LXRα and LXRβ in macrophages attenuates the potentiation of ALOX15 expression by concomitant stimulation of ACs or T09 and IL-13. Collectively, we identify a previously unrecognized mechanism of regulation whereby LXR integrates AC uptake to selectively shape Th2-dependent gene expression in AAMs.

Fig. 1. LXR- and SREBP-2-dependent gene expression in efferocytic macrophages.

Expression of a LXR-dependent genes ABCA1, ABCG1, SMPDL3A, and MERTK in primary human monocyte-derived naive macrophages cocultured with apoptotic Jurkat cells or stimulated with T09 (1 µM) for 3 h and b SREBP-2-dependent genes HMGCR, LDLR, CYP51A1, and DHCR24 in naive macrophages cocultured with apoptotic Jurkat cells for 3 h. c Cellular mass, d total cholesterol, e sterol intermediates, and f cholesterol-derived oxysterols in naive macrophages and macrophages following 3-h coculture with apoptotic Jurkat cells. g Schematic representation of the Bloch and Kandutsch–Russell pathways for the enzymatic conversion of squalene to cholesterol. Potential sites of crossover from Bloch to Kandutsch–Russell pathway are indicated by broken gray arrows. Intermediates increased in efferocytic macrophages are shown in red. Data are presented as mean ± SE from at least four independent experiments. Statistical analysis was performed using one sample t-test for a and b and two-tailed Student’s t test for c–f (**P < 0.01 and ***P < 0.001 vs control macrophages).

Fig. 2. Exogenous and endogenous sterol intermediates reciprocally regulate LXR- and SREBP-2-dependent gene expression.

Expression of a LXR-dependent genes ABCA1, ABCG1, and SMPDL3A, and b SREBP-2-dependent genes HMGCR, LDLR, and CYP51A1 in human monocyte-derived naive macrophages treated with desmosterol (2.5, 5, and 10 µM) for 24 h. c Action of triparanol, a pharmacological inhibitor of 24-dehydrocholesterol reductase, which has been shown to increase endogenous levels of desmosterol. d Gene expression of ABCG1 in macrophages treated with triparanol (5 and 10 µM) with (inactive cholesterol biosynthesis) and without (active cholesterol biosynthesis) serum for 24 h. e Expression of LXR-dependent genes ABCA1, ABCG1, and SMPDL3A in naive macrophages cotreated with triparanol (10 µM) and ACs for 3 h. Data are presented as mean ± SE from at least three independent experiments. Statistical analysis was performed using one sample t-test for a and b (*P < 0.05 vs 0 µM desmosterol), and d (*P < 0.05 vs 0 µM triparanol), and one-way ANOVA with Bonferroni post hoc means comparisons for e (*P < 0.05 vs ACs; ^#P < 0.05 vs untreated control).

Fig. 3. Identification of Th2 cytokine-dependent genes potentiated by LXR.

a Schematic representation of treatment protocol. Primary human monocyte-derived naive macrophages (hMDMФs) were pulsed with T09 (1 µM) for 3 h. Culture media was aspirated and cells washed with PBS then stimulated with Th2 cytokines IL-4 (5 ng/ml) or IL-13 (10 ng/ml) for 24 h before performing RNA-seq analysis. Heat map displaying 25 strongest upregulated genes as count-per-million (CPM) following b 24-h stimulation with IL-13 or IL-4 and c 3-h pulse with T09. Dashed gray lines connect corresponding genes. Heat map displaying d the impact of T09 on the expression of Th2 cytokine-dependent genes associated with the AAM transcriptional profile as mean CPM and e the expression of Th2 cytokine-dependent genes ALOX15, IL1RN, CIITA, XXYLT1, and LILRB1 potentiated by LXR activation as mean CPM. Validation of f ALOX15, g IL1RN, h CIITA, i XXYLT1, and j LILRB1 gene expression in naive macrophages pulsed with T09 for 3 h followed by stimulation with IL-4 or IL-13 for 24 h by real-time PCR. Data presented in b–e are from hMDMФs derived from three individual donors. Data presented in f–j are mean ± SE from at least three independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc means comparisons (*P < 0.05; ^#P < 0.05 vs untreated control).

Fig. 4. ALOX15 is modulated by LXR.

a Representative western analysis of ALOX15 expression in macrophages pulsed with T09 for 3 h followed by stimulation with IL-13 (10 ng/ml) for 48 h (left panel), and corresponding densitometric analysis from three independent experiments (right panel); *P < 0.05 using one sample t-test. b Formation of AA-, EPA-, and DHA-derived lipid mediators in macrophages pulsed with T09 for 3 h followed by stimulation with IL-13 (10 ng/ml) for 48 h. Data are presented as mean ± SE from four independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc means comparisons (*P < 0.05; ns = not significant).

Fig. 5. Engulfment of apoptotic cells potentiates IL-13-dependent ALOX15 expression.

ALOX15 a gene expression in macrophages cocultured with ACs for 3 h followed by stimulation with IL-13 (10 ng/ml) for 24 h and b protein expression in macrophages cocultured with ACs for 3 h followed by stimulation with IL-13 (5 and 10 ng/ml) for 48 h. c IL1RN, d CIITA, e XXYLT1, and f LILRB1 gene expression in macrophages cocultured with ACs for 3 h followed by stimulation with IL-13 (10 ng/ml) for 24 h. g ALOX15, IL1RN, CIITA, XXYLT1, and LILRB1 gene expression in macrophages pretreated with desmosterol (10 µM) for 16 h then cotreated with desmosterol (10 µM) and IL-13 (10 ng/ml) for 24 h. Data in a and c–g are presented as mean ± SE from at least three independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc means comparisons (*P < 0.05 vs untreated control; ^#P < 0.05 vs ACs or desmosterol; ^§P < 0.05 vs IL-13).

Fig. 6. Gene expression in LXR knockdown macrophages.

a Gene expression of LXRα and LXRβ in naive macrophages transfected with control or dual LXRα/β siRNAs. b Expression of LXR-dependent genes ABCG1 and SMPDL3A in control and dual LXRα/β knockdown macrophages cocultured with ACs for 3 h. c Gene expression of ALOX15 in control and dual LXRα/β knockdown macrophages pulsed with T09 (1 µM) for 3 h followed by 24-h stimulation with IL-13 (10 ng/ml). d Gene expression of ALOX15 and IL1RN in control and dual LXRα/β knockdown macrophages cocultured with ACs for 3 h followed by stimulation with IL-13 (10 ng/ml) for 24 h. Data are presented as mean ± SE from at least three independent experiments. Statistical analysis was performed with two-tailed Student’s t test (*P < 0.05) in a, one sample t-test (*P < 0.05) in b, and both two-tailed Student’s t test (*P < 0.05) and one sample t-test (*P < 0.05) in c and d. Statistical analysis was performed with one sample t-test (*P < 0.05), Student’s t test (^#P < 0.05), and one-way ANOVA with Bonferroni post hoc means comparisons (^§P < 0.05 vs untreated siRNA-transfected; ns = not significant).

Fig. 7. Sterols in apoptotic cells.

a Total cholesterol, b sterol intermediates lathosterol lanosterol, DHL (dihydrolanosterol), and desmosterol, and c cholesterol-derived oxysterols in Jurkat cells treated with or without staurosporine (0.5 µM) in serum-free media for 0, 3, and 6 h (7-ketoC, 7-ketocholesterol; 25-HC, 25-hydroxycholesterol; 7α-HC, 7alpha-hydroxycholesterol; 24-HC, 24-hydroxycholesterol; 4β-HC, 4beta-hydroxycholesterol; 27-HC, 27-hydroxycholesterol). Expression of d LXR-dependent genes ABCA1, ABCG1, and SMPDL3A, and e SREBP-2-dependent genes HMGCR, LDLR, and CYP51A1 in human monocyte-derived naive macrophages pretreated with U-18666A (5 µM) for 1 h then cotreated with ACs for 3 h. Data are presented as mean ± SE from at least four independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc means comparisons in a–c (*P < 0.05 vs time 0). For d and e, statistical analysis was performed with two-tailed Student’s t test (d ^#P < 0.05 vs untreated control; e *P < 0.05 vs ACs) and one sample t-test (d *P < 0.05 vs ACs and e ^#P < 0.05 vs untreated control).

Fig. 8. Mechanistic scenario for induction of ALOX15.

Accumulation of sterol intermediates derived from ACs, or synthetic LXR ligand T0901317, activate LXR to facilitate cellular sterol metabolism while simultaneously priming macrophages for enhanced expression of Th2 cytokine-dependent gene ALOX15.