Graphical abstract.

Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated up-regulation of choline transport compared to naïve macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELMα (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naïve or mice infected with the intestinal helminth Heligmosomoides polygyrus were treated with the choline kinase α inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELMα expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus, with increased egg burden. Together, these data demonstrate that choline metabolism is required for macrophage RELMα induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity.</div

Inhibiting choline metabolism drives pronounced changes in gene transcription in naïve and IL-4-polarized macrophages.

A) Schematic of bulk RNA-seq sample preparation. Heatmap of z-scores for select genes of interest. n = 3. B) Volcano plots of comparisons between Veh and HC3 or RSM in M[0] or M[IL-4]. Red dots represent genes with false discovery rate (FDR) 2 fold change (FC) 1. Select genes of interest are annotated. C-D) The top significantly up- or down-regulated genes shared in all comparisons in B) were analyzed by Enrichr-KG to identify common pathways. Schematics were created using BioRender.</p

Choline uptake and phosphorylation is required for normal IL-4 signaling and M[IL-4] phenotype.

A-D) Macrophages were treated with vehicle (DMSO) or HC3 (250 μM) for 24 h, washed, then treated with IL-4 (20 ng/mL) for 24 h. Relative expression of M[IL-4] hallmark genes Arg1, Mrc1, Chil3, or Retnla normalized to Actb and compared to M[0]. n = 3–4, representative of 3–5 experiments. Unpaired t test (*** p Retnla normalized to Actb and compared to M[0]. n = 3, representative of 1 experiment. Two-way ANOVA with Tukey’s test for multiple comparisons (* p + macrophages. n = 3, representative of >4 experiments. One-way ANOVA with Tukey’s test for multiple comparisons (* p < 0.05, ** p < 0.01). I) Macrophages were treated with vehicle (DMSO), HC3 (250 μM), or RSM-932a (5 μM) for 24 h, washed, then treated with IL-4 (20 ng/mL) for 24 h. Detection of supernatant RELMα by ELISA. n = 5 (vehicle, HC3, IL-4) or n = 2 (RSM). Unpaired t test (** p < 0.01). Schematics were created using BioRender.</p

IL-4 up-regulates choline metabolism in macrophages.

A) Schematic of lipidomics analysis. B-G) Total PC content (B), heatmap of PC species (C), SM content (D), PC(O) content (E), PC(P) content (F), or total LPC content (G) for M[0], M[IL-4], or M[LPS] expressed as nmol per 106 cells. n = 5 per polarization. Heatmap statistics are shown as fold change over the average of M[0]. One-way ANOVA with Dunnett’s test for multiple comparisons (* p 3H-choline into phospholipids over time. n = 5 per timepoint, representative of 3 experiments. Two-way ANOVA with Šídák’s test for multiple comparisons (** p 3H-choline over time in M[0] or M[IL-4]. n = 3 per timepoint, representative of 3 experiments. Linear regression F test (**** p Slc44a1 and Slc44a2 transcripts in M[0] or M[IL-4], normalized to Actb. n = 8 mice. K) Expression by Western blot of Slc44a1 and Slc44a2 in M[0] or M[IL-4]. Densitometry quantified relative to β-actin. n = 3, representative of 3 experiments. Unpaired t test (** p < 0.01). L) Expression of Slc44a1 by surface flow cytometry after different IL-4 stimulation times. Representative of 2 experiments. Schematics were created using BioRender.</p

Raw data file.

Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated upregulation of choline transport compared to naïve macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELMα (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naïve or mice infected with the intestinal helminth Heligmosomoides polygyrus were treated with the choline kinase α inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELMα expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus, with increased egg burden. Together, these data demonstrate that choline metabolism is required for macrophage RELMα induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity.</div

Choline-containing phospholipid species in polarized macrophages.

A-I) Sum of phospholipid subclasses. A) Plasmenyl-lysophosphatidylcholines (LPC(P)), B) plasmanyl-LPCs (LPC(O)), C) platelet activating factors (PAF). D) D) Heatmap of sphingomyelin (SM), plasmanyl-PAFs (PC(O)-PAF), PC(O), PC(P), LPC, LPC(O), and LPC(P). Heatmap statistics are shown as fold change over the average of M[0]. n = 5. One-way ANOVA with Dunnett’s test vs M[0] for multiple comparisons (* p (TIF)</p