LPS stimulation increases choline uptake and PC synthesis. BMDM were treated with LPS (100 ng/ml) for 48 h before measuring choline uptake. A, choline uptake was measured at the indicated times by incubating cells in a solution of KRH buffer containing 1 μCi/ml [³H]choline chloride (n = 6 separate bone marrow isolations, each performed in triplicate). B, choline uptake was measured over 10 min by incubating cells in a solution of KRH buffer containing 1 μCi/ml [³H]choline chloride and increasing amounts of nonradiolabeled “cold” choline (n = 6–8 separate bone marrow isolations, each performed in triplicate). Choline uptake was determined by measuring intracellular radioactivity, which was plotted against the amount of protein. C, BMDM were treated with LPS (100 ng/ml) for 48 h before treatment with 1 μCi/ml [³H]choline chloride in DMEM for the indicated times, and the incorporation into PC was determined (n = 6–9 separate bone marrow isolations, each performed in triplicate). Data are expressed as mean ± S.E. (error bars). The rates of choline uptake (A) were determined via linear regression, where the p value indicated represents significance between M[0] and M[LPS] cells. For choline uptake kinetics (B), data were fit to the Michaelis–Menten curve, and statistical significance is represented as follows: ****, p < 0.0001, as determined by a comparison of the curve fit using extra sum-of-squares F-test. For PC synthesis (C), statistical significance is represented as follows: *, p < 0.05; **, p < 0.01; ****, p < 0.0001 compared between treatments; ##, p < 0.01 compared with the 2-h time point determined by two-way ANOVA.

Macrophage LPS-polarization increases PC and de novo lipogenesis. BMDM were treated with or without LPS (100 ng/ml) for 48 h. HPLC-ESI-MS/MS lipidomics was used to assess total PC content (A) and PC fatty acid composition (B) (n = 3 separate bone marrow isolations). Cells treated with and without LPS were incubated in the presence of [³H]acetate to measure fatty acid synthesis, where C is the incorporation into all lipids, with the proportional incorporation of [³H]acetate-derived fatty acids into phospholipid (PL), DAG, TG, or cholesteryl ester (CE) shown to the right. Data for A–C are expressed as mean ± S.E. (error bars), where statistical significance is represented as follows: *, p < 0.05; ****, p < 0.0001 compared with M[0]-treated cells as determined by two-way ANOVA as indicated. For D, M[LPS] is shown relative to M[0], where the log₂-transformed relative difference is reflected by the size of the pie (n = 4 separate bone marrow isolations, each performed in triplicate).

Choline transporter transcript and protein expression induced by LPS. BMDM were treated with LPS (100 ng/ml) for 48 h. A, qPCR determination of the relative expression of Slc44a1, Slc44a2, Chka, Pcyt1a, and Pcyt2, which were normalized to the average expression of β-actin and Tbp (n = 4 separate bone marrow isolations, each performed in triplicate). Data are mean ± S.E. (error bars), where statistical significance is represented as follows: ****, p < 0.0001 compared with M[0] cells as determined by an unpaired two-tailed Student's t test. B, protein expression of CTL1 and CTL2 was determined and normalized to β-actin (n = 3 separate bone marrow isolations, each run in duplicate). Data are mean ± S.E. of the densitometry quantification (ImageJ), and the blots are representative images of the biological replicates, where statistical significance is represented as follows: **, p < 0.01 compared with M[0] cells as determined by an unpaired two-tailed Student's t test.

Chronic pharmacological or CTL1-specific antibody inhibition lowers choline uptake and PC levels. BMDM were incubated for 48 h in the presence or absence of the choline uptake inhibitor HC3 (250 μm), where DMSO was used as a vehicle control or with a primary CTL1 antibody, where an isotype control IgG antibody was used (both at 1:250 dilution). A, acute [³H]choline uptake was assessed over 10 min (n = 4 separate isolations, each performed in triplicate). B, chronic choline uptake inhibition was accompanied by chronic [³H]choline incorporation into PC (n = 4 separate isolations, each performed in triplicate). Data are mean ± S.E. (error bars), where statistical significance is represented as follows: ***, p < 0.001 compared with control treatment as indicated and assessed by one-way ANOVA.

Inhibiting choline uptake alters LPS-induced cytokine secretion. Cells were incubated in the presence or absence of HC3 (250 μm) or CTL1-Ab (1:250) for 48 h. The medium was removed, and cells were stimulated with 100 ng/ml LPS for 6 h (with inhibitor treatments replenished), and from the supernatant, TNFα (A), IL-6 (B), and IL-10 secretion (C) were determined (n = 5 separate isolations, each performed in triplicate). Data are mean ± S.E. (error bars), where statistical significance is represented as follows: ****, p < 0.0001 compared with LPS-stimulated control treatment, as indicated, assessed by two-way ANOVA.

Inhibiting choline uptake causes accumulation and redirection of DAG. BMDM were pulsed with [³H]glycerol for 48 h in the presence or absence of choline uptake inhibitor HC3 (250 μm) or the CTL1-Ab (1:250). Lipids were then extracted and separated via TLC to determine radioactivity of DAG (A), TG (B), and PC (C) (n = at least 4 separate isolations). D, qPCR determination of the relative expressions of Dgat1 and Dgat2 after 48-h treatment, as indicated, which were normalized to the average expression of β-actin (n = 4 separate bone marrow isolations, each performed in triplicate). E, representative images of the maximum intensity projection confocal images of cells incubated with DMSO or HC3 (250 μm) for 48 h and stained with DAPI or Nile Red. Scale bars, 10 μm. Corner insets are enlarged from the yellow boxes. Data are mean ± S.E. (error bars), where statistical significance for A–C is represented as follows: **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 compared with control treatment as indicated, assessed by one-way ANOVA. Statistical significance for D is represented as follows: *, p < 0.05; **, p < 0.01 compared with DMSO control, determined by Student's t test.

Inhibiting choline uptake is associated with higher PKC activity. Choline uptake was inhibited in BMDM for 48 h using HC3 (250 μm). Cell lysate was collected and immunoblotted using a PKC substrate motif antibody. Equal loading was confirmed by using β-actin, run on a duplicate gel (n = 4 separate isolations, each performed in duplicate). Blots are representative images of the biological replicates.

Extracellular choline or PKC inhibition rescues cytokine secretion profile. Cells were incubated in the presence or absence of HC3 (250 μm) or the CTL1-Ab (1:250) for 48 h and co-treated with excess choline chloride (500 μm) or the PKC inhibitor BIM (20 μm). The medium was removed, and cells were stimulated with 100 ng/ml LPS for 6 h, during which time the initial treatments were replenished. Secretion of TNFα (A and D), IL-6 (B and E), and IL-10 (C and F) into the medium was determined (n = 5 separate isolations, each performed in triplicate). Data are mean ± S.E. (error bars), where statistical significance is represented as follows: ****, p < 0.0001 compared with LPS-stimulated control or control IgG treatment; #, p < 0.05; ##, p < 0.01; ###, p < 0.001 compared with HC3, as indicated, assessed by one-way ANOVA.

PC levels in the presence of choline or PKC inhibitor. A, cells were incubated with or without HC3 (250 μm), excess choline chloride (500 μm), or BIM (20 μm) for 48 h to assess PC levels (n = 4 separate isolations performed in duplicate). B, cells were treated with the CTL1-Ab (1:250), excess choline (500 μm), or BIM (20 μm) to assess PC levels (n = 4 separate isolations performed in duplicate). Data are mean ± S.E. (error bars), where statistical significance is represented as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with indicated control; ##, p < 0.01 compared with HC3 (A) group, as indicated, assessed by one-way ANOVA.