IL-4 up-regulates choline metabolism in macrophages.

A) Saturation curves showing rate of 3H-choline uptake at increasing concentrations of unlabeled choline. n = 8. Michaelis-Menten least squares fit regression (**** p 3H-choline uptake by HC3. n = 4 (M[0]) or 5 (M[IL-4]). Four parameter log(inhibitor) vs. response regression F test (** p (TIF)</p

Reactome pathway analysis.

A-D) Reactome pathway analysis of genes up-regulated by HC3 (A) or RSM (C) in M[IL-4] or down-regulated by HC3 (B) or RSM (D), sorted by gene count enrichment for each Reactome pathway. (TIF)</p

<i>In vivo</i> choline kinase inhibition in naïve mice and primary intestinal helminth infection alters peritoneal cell populations and macrophage alternative activation.

A) UMAP plots of peritoneal cells (PECs) from naïve and H. polygyrus-infected mice treated with vehicle or RSM-932a. See S6A Fig for population gating. B-G) Enumeration of B) total PECs, C) eosinophils, D) monocytes, E) B-1 cells, F) neutrophils, G) peritoneal macrophages among live PECs. n = 3–4 (naïve) or n = 5 (infected). Two-way ANOVA with Šídák’s test for multiple comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). H-J) CD206, I) CD86, or J) PD-L1 expression (gMFI) on peritoneal macrophages. n = 3–4 (naïve) or n = 5 (infected). Two-way ANOVA with Šídák’s test for multiple comparisons (* p < 0.05), representative of 2 experiments.</p

Inhibiting choline metabolism impairs mitochondrial structure and function.

A) Heatmap of z-scores for select mitochondrially-encoded genes. B) Western Blot of electron transport chain complexes I to V in macrophages treated with vehicle (DMSO), HC3 (250 μM), or RSM (1 or 5 μM). Total protein by trichloroethanol (TCE) staining shown for normalization. Representative blots of n = 3. C-I) Mito Stress Test assay of extracellular flux with sequential treatments of 1.5 μM oligomycin, 14 μM BAM15, and 1 μM rotenone/1 μM antimycin A/Hoechst 33342. Oxygen consumption rate (OCR) of M[0] (C) or M[IL-4] (E) treated with DMSO (Veh), HC3 (250 μM), or different concentrations of RSM. Extracellular acidification rate (ECAR) in M[0] (D) or M[IL-4] (E). Derived parameters of basal respiration (G), spare respiratory capacity (H), or non-mitochondrial respiration (I) from (C) and (E). Measurements (n = 3 in triplicate) were normalized to an arbitrary cell factor determined by Hoechst 33342 nuclei counts. One-way ANOVA with Šídák’s test for multiple comparisons (*** p OXPHOS) or glycolysis (ATPGlyco). Measurements (n = 3 in triplicate) were normalized per 103 cells. Mixed-effects analysis with Tukey’s test for multiple comparisons (ATPOXPHOS: ## p Glyco: * p < 0.05, *** p < 0.001, **** p < 0.0001). K) Transmission electron micrographs of macrophages treated with vehicle (DMSO) or RSM (5 μM) in M[IL-4]. Scale bars represent 2 μM (left) or 0.5 μM (right insets). White arrows indicate healthy mitochondria with intact cristae, orange arrows indicate mitochondria with aberrant membrane structure or absent cristae. Representative images from n = 3 and at least 10 cells imaged per condition. No cells with intact mitochondria were observed with RSM treatment.</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

<i>In vivo</i> choline kinase inhibition remodels peritoneal cell populations and impairs RELMα expression.

A) Schematic of 7-day in vivo choline kinase inhibition. Mice were treated intraperitoneally with vehicle (40% DMSO in PBS) or RSM-932a (3 mg/kg) every other day for 6 days and sacrificed on day 7. B) UMAP of peritoneal cell populations. n = 9–10, representing 2 independent experiments. C) Intracellular RELMα expression in live CD11b+F4/80hi large (LPM) and CD11b+F4/80lo small (SPM) peritoneal macrophages. Two-way ANOVA with Šídák’s test for multiple comparisons (* p +F4/80hi LPM and CD11b+F4/80lo SPM. Two-way ANOVA with Šídák’s test for multiple comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001). F) Intracellular RELMα expression in LPM and SPM isolated from peritoneal cavity aggregates or suspended lavage cells. Two-way ANOVA with Šídák’s test for multiple comparisons (* p < 0.05, **** p < 0.0001). Schematics were created using BioRender.</p

<i>In vivo</i> choline kinase inhibition during secondary intestinal helminth infection alters peritoneal cell populations and macrophage alternative activation.

A-D) Enumeration of A) B-1 cells, B) monocytes, C) neutrophils, D) peritoneal macrophages among live PECs. n = 4–5. Unpaired t test (** p (TIF)</p

Mitochondrial changes induced by inhibiting choline metabolism.

A) Densitometry of complex III and I from Fig 4B. Two-way ANOVA with Dunnett’s test for multiple comparisons. B) Bioenergetics analysis of ATP produced through oxidative phosphorylation (ATPOXPHOS) or glycolysis (ATPGlyco). Macrophages were treated for 1 h or 5 min with RSM (0.2, 1, or 5 μM) prior to Mito Stress Test assay as in Fig 4C–4F. Measurements (n = 3 in triplicate) were normalized per 103 cells. Mixed-effects analysis with Tukey’s test for multiple comparisons (ATPOXPHOS: # p Glyco: ** p (TIF)</p

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