Lipin-1 restrains macrophage lipid synthesis to promote inflammation resolution

Abstract

Macrophages are critical to maintaining and restoring tissue homeostasis during inflammation. The lipid metabolic state of macrophages influences their function, but a deeper understanding of how lipid metabolism is regulated in pro-resolving macrophage responses is needed. Lipin-1 is a phosphatidic acid phosphatase with a transcriptional coregulatory activity (TC) that regulates lipid metabolism. We previously demonstrated that lipin-1 supports pro-resolving macrophage responses, and here, myeloid-associated lipin-1 is required for inflammation resolution, yet how lipin-1-regulated cellular mechanisms promote macrophage pro-resolution responses is unknown. We demonstrated that the loss of lipin-1 in macrophages led to increased free fatty acid, neutral lipid, and ceramide content and increased phosphorylation of acetyl-CoA carboxylase. The inhibition of the first step of lipid synthesis and transport of citrate from the mitochondria in macrophages reduced lipid content and restored efferocytosis and inflammation resolution in lipin-1^mKO macrophages and mice. Our findings suggest macrophage-associated lipin-1 restrains lipid synthesis, promoting pro-resolving macrophage function in response to pro-resolving stimuli.

Keywords: Lipin-1; beta-oxidation; citrate; citrate carrier; efferocytosis; fatty acid; inflammation resolution; lipid metabolism; lipid synthesis; macrophage; metabolism; pro-resolving.

Fig. 1.. Loss of myeloid lipin-1 delays inflammation resolution.

(A) Mice were subjected to a zymosan challenge (0.1mg/mouse). PMNs and macrophages were quantified from the peritoneal cavity by flow cytometry. (B) Total number of cells isolated from the peritoneal cavity. (C) Total number of PMNs isolated from the peritoneal cavity. Resolution interval (Ri) was determined. (D) Total number of Macrophages isolated from the peritoneal cavity. Illustration in (A) was created using BioRender.com. A minimum of three mice per group per time point were analyzed. N ≥3 mice per group per time point. Values are means ± SEM. Unpaired two-tailed T-tests were performed between groups at each time point. *= p≤0.05.

Fig. 2.. Lipin-1 promotes pro-resolution phenotype in vivo.

Mice were challenged with 0.1mg zymosan, and six days later, peritoneal cells were isolated by lavage. Isolated cells were stained for flow cytometric analysis. (A) The number and ratio of CD11b+ F4/80+ lin- cells in the peritoneal cavity. (B-C) Median fluorescent Intensity of metabolic and inflammatory markers of CD11b+ F4/80+ lin- cells. (D) UMAP and phenograph clustering of macrophages and those enriched in wild-type (orange) and lipin-1mKO (purple) mice using metabolic markers. (E) heat map of Percent population changes of phenoclusters between wild-type and lipin-1mKO mice and Min-Max heat map of metabolic and inflammatory markers from identified phenoclusters. (F) The number and ratio of CD11b+ F4/80+ lin- cells by phenocluster type. (G-H) Median fluorescent Intensity of metabolic and inflammatory markers of CD11b+ F4/80+ lin- cells from phenocluster 6 in wild-type mice (orange) and phenoclusters 9, 4, & 2 from lipin-1^mKO (purple) mice. N=3 mice per group. Dots represent individual mice; lines are means. Unpaired Two-tailed T-test was performed between groups to define significance *= p≤0.05 Abbreviations: Glut 1: glucose transporter, PKM: Pyruvate Kinase M2, G6PD: Glucose-6-phosphate dehydrogenase, ACC1: Acetyl CoA carboxylase 1, CPT1A: mitochondria fatty acid importer, SDHA: Succinate dehydrogenase, CytC: Cytochrome C, PDL: Programmed Death-Ligand

Fig. 3.. Loss of lipin-1 results in an increase in fatty acids, neutral lipids, and ceramides.

Lipids harvested from IL-4-stimulated (40ng/ml, 4 hours) BMDMs were processed via LC–MS for lipidomics analysis. (A) PCA analysis of z-score values shows the spatial distribution of lipids within each condition. (B) Heat map of most-changed lipid species relative to untreated cells (n=6). A 3% FDR correction of student’s t-test analysis between fold change adjusted IL-4 stimulated WT and KO values was used to populate lipids in panel B (C) Heat map of lipid species as a family (n=6). Lipids that are represented by family in (C) include all respective species that passed the 3000 units cut-off, regardless of statistical significance. (D) Quantified signals of lipid species from the ceramide and dihydroceramide family (n=6). (E) Quantification of lipid families in C.(F) Nile red staining images of IL-4 stimulated (40ng/ml, 4 hours) BMDMs. Heatmaps were made of Log2 Fold Change Analysis from WT untreated. Significance was determined by one-way ANOVA *= p≤0.05, **= p≤0.01, ***= p≤0.001, and ****= p≤0.001. Lipid abbreviations: FA: Fatty acids, DAG: Diglycerides, TAG: Triglycerides, DihCer: Dihydroceramide, Cer: Ceramide, GlcCer Glucosylceramide, SM: Sphingomyelin, MAG: Monoacylglycerol, LPC: Lysophosphatidylcholine, LPE: Lysophosphatidylethanolamine, LPI: Lysophosphatidylinositol, LPS: Lysophosphatidylserine, LPG: Phosphatidylglycerol, PE: Phosphatidylethanolamine, PC: Phosphatidylcholines, PG: Phosphatidylglycerol, PS: Phosphatidylserine, PI: Phosphatidylinositol, PE O: Ether-linked Phosphatidylethanolamine, PE P: Phosphatidylethanolamine plasmalogen.

Fig. 4.. Loss of lipin-1 during IL-4 stimulation results in a failure to restrain fatty acid biosynthesis.

(A) Illustration of the de novo fatty acid biosynthesis pathway (B-C) Protein was isolated from IL-4-stimulated (40ng/ml, 4 hours) whole cells, and protein abundance of fatty acid biosynthesis enzymes and proteins was quantified by Western blot analysis (n>3). (D) Representative western blot images and quantified protein abundance of histone proteins and post-translational modifications (n=X). Illustration in (A) was created using BioRender.com. Bars represent the standard error of the mean (± SEM). Significance was determined by one-way ANOVA (fold change) and paired student’s t-test (ratio). *= p≤0.05, **= p≤0.01.

Fig. 5.. Inhibition of the citrate carrier and de novo ceramide biosynthesis restores efferocytic capacity in lipin-1 ko macrophages.

(A) BMDMs from lipin-1^mKO mice and littermate controls were stimulated with IL-4 for 6 hours and then challenged with CFSE-labeled apoptotic cells. (B) Illustration of the de novo fatty acid biosynthesis pathway and checkpoints of inhibition. (C) BMDMs were treated with 10μM inhibitors (FAS: C75, ACC: Cpd9, CTP: CTPi) and DMSO Vehicle (VEH) for 12 hours and subsequently cotreated with 40ng/ml IL4 and 10μM of respective inhibitors for 6 hours. Images were taken at 20x and zoomed at 1.5x to generate representative images. The experiment was done twice, each time with 3 unique pairs of individual WT and FKO (full KO) BMDMs (n= 6). At least 3 random images of each group were taken, quantified (C), and grouped to give individual dots. Individual experiments incorporated all inhibitors, so each inhibitor group had the same vehicle control. (D) BMDMs were treated with increasing concentrations of SCT (1mM, 3mM, 6mM) for 2 hours before co-treatment with IL-4 for 6 hours. In vitro efferocytosis was carried out (E). In vitro efferocytosis with 10μM Myriocin. BMDMs were treated with Myriocin for 2 hours and cotreated with Myriocin and IL-4 for 6 hours before the AC challenge. Illustration in (B) was created using BioRender.com. Bars represent the standard error of the mean (± SEM). Significance was determined by two-way ANOVA. *= p≤0.05, **= p≤0.01, ***= p≤0.001, and ****= p≤0.001.

Fig. 6.. Inhibition of citrate carrier reduced neutral lipids levels without restoring mitochondria respiratory capacity.

(A) BMDMs isolated from lipin-1mKO mice and littermate controls were treated with CTPi for 12 hours before cotreatment with CTPi and 40ng/ml IL-4 for 4 hours. Oxygen consumption rate (OCR), and mitochondrial function parameters were analyzed via Seahorse extracellular flux analyzer. Graphed data represent mean OCR with SEM (n=4) (B) BMDMs were treated with 10μM inhibitors (FAS: C75, ACC: Cpd9, CTP: CTPi) and DMSO Vehicle (VEH) for 12hrs and subsequently cotreated with 40ng/ml IL4 and 10μM of respective inhibitors for 4 hours before Nile red staining. Images were taken at 10x to generate representative images. The experiment was done twice, each time with 2 unique pairs of individual WT and FKO (full KO) BMDMs (n=4). At least 3 random images of each group were taken, quantified, and grouped to give individual dots. Bars represent the standard error of the mean (± SEM). Significance was determined by one-way and two-way ANOVA for Seahorse data and Nile red values, respectively. *= p≤0.05, **= p≤0.01, ***= p≤0.001.

Fig. 7.. Inhibition of the citrate carrier restores inflammation resolution in lipin-1^mko mice.

(A) Lipin-1^mKO mice and littermate controls were injected with 50mg/kg CTP-2 or C75 at 12 hours and 18 hours post Zymosan injection. (B) Flow cytometry analysis of intraperitoneal lavage after acute (0.1mg/mouse, 24 hours) Zymosan injection. Illustration in (A and C) was created using BioRender.com. Bars represent the standard error of the mean (± SEM). Significance was determined by one-way ANOVA. *= p≤0.05, **= p≤0.01.