A Novel Function for 15-Lipoxygenases in Cholesterol Homeostasis and CCL17 Production in Human Macrophages

Abstract

Arachidonate 15-lipoxygenase (ALOX15) and arachidonate 15-lipoxygenase, type B (ALOX15B) catalyze the dioxygenation of polyunsaturated fatty acids and are upregulated in human alternatively activated macrophages (AAMs) induced by Th2 cytokine interleukin-4 (IL-4) and/or interleukin-13. Known primarily for roles in bioactive lipid mediator synthesis, 15-lipoxygenases (15-LOXs) have been implicated in various macrophage functions including efferocytosis and ferroptosis. Using a combination of inhibitors and siRNAs to suppress 15-LOX isoforms, we studied the role of 15-LOXs in cellular cholesterol homeostasis and immune function in naïve and AAMs. Silencing or inhibiting the 15-LOX isoforms impaired sterol regulatory element binding protein (SREBP)-2 signaling by inhibiting SREBP-2 processing into mature transcription factor and reduced SREBP-2 binding to sterol regulatory elements and subsequent target gene expression. Silencing ALOX15B reduced cellular cholesterol and the cholesterol intermediates desmosterol, lanosterol, 24,25-dihydrolanosterol, and lathosterol as well as oxysterols in IL-4-stimulated macrophages. In addition, attenuating both 15-LOX isoforms did not generally affect IL-4 gene expression but rather uniquely impacted IL-4-induced CCL17 production in an SREBP-2-dependent manner resulting in reduced T cell migration to macrophage conditioned media. In conclusion, we identified a novel role for ALOX15B, and to a lesser extent ALOX15, in cholesterol homeostasis and CCL17 production in human macrophages.

Keywords: arachidonate 15-lipoxygenase; chemokine; cholesterol; interleukin-4; lipoxygenase; macrophage; sterol regulatory element binding protein-2; type B.

Figure 1

Lipoxygenase expression in interleukin-4 (IL-4)-stimulated macrophages. (A) Flow cytometric analysis of CD206, CD86, CD163, and CD80 (B) and mRNA expression of 5-lipoxygenase (ALOX5), 12-lipoxygenase (ALOX12), 15-lipoxygenase (ALOX15), and 15-lipoxygenase, type B (ALOX15B) in primary human macrophages treated with 20 ng/ml IL-4 for 48 h. P-values were calculated using two-tailed Student’s t-test. (C) Western analysis of ALOX15, ALOX15B, and ALOX5 expression in macrophages treated for indicated times with IL-4. (D) LC-MS/MS analysis of arachidonic acid metabolites 5-HETE, 12-HETE, 15-HETE, and linoleic acid (LA) metabolite 13-HODE in macrophages treated for 48 h with IL-4. P-values were calculated using two-tailed Student’s t-test. (E,F) Western analysis of ALOX15 and ALOX15B expression in macrophages transfected with control and ALOX15 (D) or ALOX15B (E) siRNAs 24 h prior to treatment with IL-4 for 48 h. (G) LC-MS/MS analysis of arachidonic acid metabolites 5-HETE, 12-HETE, 15-HETE, and LA metabolite 13-HODE in macrophages transfected with control, ALOX15, or ALOX15B siRNAs 24 h prior to treatment with IL-4 for 48 h. P-values were calculated using one-way ANOVA with Bonferroni post hoc means comparisons with significance level set at 0.05. ***P < 0.001. Data are mean values ± SE of at least three independent experiments.

Figure 2

Knockdown of arachidonate 15-lipoxygenase (ALOX15) and arachidonate 15-lipoxygenase, type B (ALOX15B) inhibits sterol regulatory element binding protein (SREBP)-2 activation. (A) Representative Western analysis of SREBP-2 and mature N-terminal SREBP-2 (mSREBP-2) in macrophages transfected with control, ALOX15, or ALOX15B siRNAs 24 h prior to treatment with interleukin-4 (IL-4) for 48 h. (B–D) mRNA expression of (B,C) low density lipoprotein receptor (LDLR), mevalonate kinase (MVK), acetoacetyl-CoA synthetase (AACS), acetyl-CoA acetyltransferase 2 (ACAT2), insulin-induced gene 1 (INSIG1) (D) and SREBP-1c, apolipoprotein E (APOE), liver X receptor (LXR)α, myosin regulatory light chain interacting protein (MYLIP), ABCG1, ABCA1 in macrophages transfected with control and ALOX15 (B,D) or ALOX15B (C,D) siRNAs 24 h prior to treatment with IL-4 for 48 h. P-values were calculated using two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Data are mean values ± SE of at least three independent experiments.

Figure 3

Sterol levels in interleukin-4 (IL-4)-stimulated macrophages. (A,B) Total (A) and free (B) cholesterol in macrophages transfected with control, arachidonate 15-lipoxygenase (ALOX15), or arachidonate 15-lipoxygenase, type B (ALOX15B) siRNAs 24 h prior to treatment with IL-4 for 48 h. P-values were calculated using one-way ANOVA with Bonferroni post hoc means comparisons with significance level set at 0.05. (C) Total cholesterol in macrophages treated with ML351 or PD146176 for 24 h. P-values were calculated using two-tailed Student’s t-test. (D) Cholesterol and non-cholesterol sterols (E) and oxysterols in macrophages transfected with control, ALOX15, or ALOX15B siRNAs 24 h prior to treatment with IL-4 for 48 h measured by gas chromatography. P-values were calculated using one-way ANOVA with Bonferroni post hoc means comparisons with significance level set at 0.05. *P < 0.05. Data are mean values ± SE of at least three independent experiments.

Figure 4

Inhibiting 15-lipoxygenase activity reduces sterol regulatory element binding protein (SREBP)-2 binding to sterol regulatory elements. (A–C) Chromatin immunoprecipitation (ChIP) analysis of SREBP-2 binding to promoter of (A) 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), (B) low density lipoprotein receptor (LDLR), and (C) mevalonate kinase (MVK) in untreated or macrophages stimulated with interleukin-4 (IL-4) and ML351 or IL-4 alone for 16 h. (D–F) mRNA expression of (D) HMGCR, (E) LDLR, and (F) MVK in macrophages stimulated with IL-4 and ML351 or IL-4 alone in a time-dependent manner. P-values were calculated using one-way ANOVA with Bonferroni post hoc means comparisons with significance level set at 0.05. *P < 0.05. Data are mean values ± SE of four independent experiments.

Figure 5

Chemokine modulation by 15-LOX isoforms in interleukin-4 (IL-4)-stimulated macrophages. (A) mRNA expression of CCL8, CCL13, CCL17, CCL18, CCL22, and CCL24 in primary human macrophages treated with 20 ng/ml IL-4 for 48 h. (B,C) Chemokine mRNA expression in macrophages transfected with control and arachidonate 15-lipoxygenase (ALOX15) (B) or arachidonate 15-lipoxygenase, type B (ALOX15B) (C) siRNAs 24 h prior to treatment with IL-4 for 48 h. (D) CCL17 protein concentrations in culture medium of macrophages transfected with control, ALOX15, or ALOX15B siRNAs 24 h prior to treatment with IL-4 for 48 h. (E,F) CCL17 mRNA expression (E) and protein concentrations (F) from macrophages treated with either ML351 (10 µM) or PD146176 (10 µM) for the final 24 h post IL-4 stimulation. P-values were calculated using one-way ANOVA with Bonferroni post hoc means comparisons with significance level set at 0.05. *P < 0.05, **P < 0.01, ***P < 0.001. Data are mean values ± SE of at least three independent experiments.

Figure 6

Inhibiting sterol regulatory element binding protein (SREBP)-2 activation reduces CCL17 expression. (A) mRNA expression of CCL17, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and low density lipoprotein receptor (LDLR) in macrophages treated with indicated concentrations of 25-HC and interleukin-4 (IL-4) for 24 h. (B) mRNA expression of CCL17, SREBP2, HMGCR, and LDLR in macrophages transfected with control or SREBP2 siRNA 24 h prior to treatment with IL-4 for 48 h. P-values were calculated using one-way ANOVA with Bonferroni post hoc means comparisons with significance level set at 0.05. *P < 0.05, **P < 0.01, ***P < 0.001. Data are mean values ± SE of at least three independent experiments.

Figure 7

Inducing sterol regulatory element binding protein (SREBP)-2 activation enhances CCL17 expression. (A) mRNA expression of CCL17, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and low density lipoprotein receptor (LDLR) in macrophages cultured overnight in serum-free RPMI containing MβCD (1 mM) then treated with interleukin-4 (IL-4) in serum-free media for 6 h. (B) mRNA expression of CCL17, HMGCR, and LDLR in macrophages treated with IL-4 and Bafilomycin A1 (100 nM) for 24 h. P-values were calculated using one-way ANOVA with Bonferroni post hoc means comparisons with significance level set at 0.05. *P < 0.05, **P < 0.01, ***P < 0.001. Data are mean values ± SE of at least three independent experiments.

Figure 8

Migration of HUT78 cells to macrophage conditioned media (CM). (A) Migration of HUT78 cells to serum-free media or CM from untreated or interleukin-4 (IL-4)-stimulated macrophages co-incubated with or without PD146176 and ML351. (B) Migration of HUT78 cells pre-treated with or without CC chemokine receptor 4 (CCR4) antagonist to CM from untreated or IL-4-stimulated human primary macrophages. P-values were calculated using one-way ANOVA with Bonferroni post hoc means comparisons with significance level set at 0.05. **P < 0.01. Data are mean values ± SE of at least three independent experiments.

Figure 9

Expression of CCL17, arachidonate 15-lipoxygenase, type B (ALOX15B), and sterol regulatory element binding protein (SREBP)-2 target genes in asthmatic subjects. (A) CCL17 and ALOX15B expression in bronchial alveolar lavage (BAL) cells isolated from healthy controls (n = 12), moderate asthmatics (n = 28), and severe asthmatics (n = 41). P-values were calculated using one-way ANOVA with Bonferroni post hoc means comparisons with significance level set at 0.05. *P < 0.05, ***P < 0.001. (B) Expression of SREBP-2 target gene lanosterol synthase (LSS), low density lipoprotein receptor (LDLR), 7-dehydrocholesterol reductase (DHCR7), SREBP cleavage-activating protein (SCAP), 24-dehydrocholesterol reductase (DHCR24), SREBP2, insulin-induced gene 1 (INSIG1), phosphomevalonate kinase (PMVK), acetoacetyl-CoA synthetase (AACS), mevalonate kinase (MVK), 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) in BAL cells isolated from control subjects and severe asthmatics. P-values were calculated using two-tailed Student’s t-test. *P < 0.05. (C) Pairwise correlations of gene expression in BAL cells isolated from severe asthmatics. The heat map indicates pairwise correlation strength (Spearman’s ρ) between ALOX15B and SREBP-2 target genes.