Macrophages play a critical role in obesity-associated chronic inflammation and disorders. However, the molecular mechanisms underlying the response of macrophages to elevated fatty acids (FAs) and their contribution to metabolic inflammation in obesity remain to be fully elucidated. In this article, we report a new mechanism by which dietary FAs, in particular, saturated FAs (sFAs), are able to directly trigger macrophage cell death. We demonstrated that excess sFAs, but not unsaturated FAs, induced the production of cytotoxic ceramides (Cers) in macrophage cell lines. Most importantly, expression of adipose FA binding protein (A-FABP) in macrophages facilitated metabolism of excess sFAs for Cer synthesis. Inhibition or deficiency of A-FABP in macrophage cell lines decreased sFA-induced Cer production, thereby resulting in reduced cell death. Furthermore, we validated the role of A-FABP in promoting sFA-induced macrophage cell death with primary bone marrow–derived macrophages and high-fat diet–induced obese mice. Altogether, our data reveal that excess dietary sFAs may serve as direct triggers in induction of Cer production and macrophage cell death through elevated expression of A-FABP, thus establishing A-FABP as a new molecular sensor in triggering macrophage-associated sterile inflammation in obesity.

Because of over nutrition and less energy expenditure, obesity in humans has reached alarming proportions (1). According to the Centers for Disease Control and Prevention, ∼34.9% of adults and 17% of children and adolescents in the United States are obese. It is now clear that obesity represents a major risk factor for many comorbid conditions, such as cardiovascular diseases, type 2 diabetes, certain types of cancers, among others (2, 3). Although impaired immunity has been linked to the development of obesity-related conditions (4, 5), detailed cellular and molecular mechanisms of how obesity causes dysfunction of immunity remain largely unknown.

As one major arm of innate immunity, macrophages play a critical role in obesity-induced chronic inflammation and disorders (6, 7). For example, accumulation of lipid-laden macrophages in blood vessels is central to the pathogenesis of atherosclerosis (8). Obesity-induced insulin resistance and type 2 diabetes are attributed to alterations of polarization and function of macrophages from the M2 to the M1 phenotype (9). Our recent studies also have shown that consumption of a high-fat diet induces infiltration of CD11c+ macrophages in the skin, which promotes chronic skin inflammation in mice (10). Although reports demonstrate that obesity-imprinted macrophages contribute to metabolic inflammation through the production of various proinflammatory mediators, the underlying molecular mechanisms by which macrophages respond to different obese factors needs to be further investigated.

Obesity is closely associated with elevated levels of fatty acids (FAs) in the circulation and various tissues (11). Plasma-free FAs (also called nonesterified FAs) are usually bound to plasma albumin, which usually ranges from 200 to 400 μM in healthy people, but from 400 to 800 μM in obese individuals and diabetic patients (12, 13). Although it seems clear that saturated FAs (sFA [e.g., palmitic acid (PA) and stearic acid (SA)]) are more lipotoxic than unsaturated FAs (uFAs [e.g., oleic acid (OA) and linoleic acid (LA)]) to various types of cells, whether and how they are transported and metabolized in macrophages to induce chronic inflammation in obesity is unclear (1416). As lipid chaperones inside cells, FA binding proteins (FABPs) facilitate lipid transport and distribution, and coordinate their responses (17, 18). The two major FABP members expressed in immune cells are adipose FABP (A-FABP) and epidermal FABP (E-FABP). Whereas E-FABP is expressed in various immune cell types, including T cells, NK cells, B cells, and macrophages (1921), A-FABP expression is restricted to APCs, in particular in macrophages (17, 22). This suggests a unique role of A-FABP in regulating lipid traffic and metabolism in macrophages. Our previous studies demonstrated that FABP expression in macrophages is critical in promotion of autoimmune inflammation and in the enhancement of tumor immunosurveillance in lean subjects (23, 24). It is therefore of great interest to investigate whether and how FABPs regulate macrophage responses and cell fate in response to excess lipid signals in the setting of obesity.

In this study, to mimic circulating excess FAs in obese conditions as reported previously (25, 26), we exposed macrophages to different concentrations of dietary FAs and observed macrophage responses and cell death. Our data indicate that macrophages responded differently to saturated versus uFAs. Whereas uFAs were metabolized and stored as lipid droplets in macrophages, sFAs were prone to produce ceramides (Cers), leading to macrophage cell death. Most importantly, we identify A-FABP as a new molecular sensor mediating sFA-induced Cer production and cell death in both immortalized and primary macrophages.

A-FABP–deficient mice (A-FABP−/−) and their wild type (WT) littermates (C57BL/6 background) were bred and housed in the animal facility of the Hormel Institute, University of Minnesota or in the animal facility of the University of Louisville. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Minnesota and the University of Louisville. After weaning, male mice were fed ad libitum either a high-fat diet (60% fat) or a control low-fat diet (10%) (Research Diets) for 6 mo before they were sacrificed for analysis of macrophage phenotype in the peripheral blood.

Monoclonal anti-Cer Ab (mouse IgM, clone MID 15B4), reactive oxygen species (ROS) inhibitors 4-amino-2,4-pyrrolidine-dicarboxylic acid (APDC) and butylated hydroxyanisole (BHA), and triacsin C were purchased from Sigma. All caspase inhibitors were from R&D Systems and Biovision. Cer synthesis inhibitor fumonisin B1 (FB1) and myriocin (a serine palmitoyltransferase) were from Cayman Chemical. BODIPY-C16 FA was purchased from Thermo Fisher Scientific. All flow cytometric Abs were purchased from BioLegend. All FA sodium salts were from Nu-Chek Prep. PA (5 mM), SA (5 mM), OA (5 mM), LA (5 mM), and ω-3 eicosapentaenoic acid were prepared with 2 mM of endotoxin-free BSA in PBS, sonicated until dissolved, and filtered through 0.2-μM sterile filter.

Primary macrophages were differentiated from either M-CSF–induced bone marrow–derived macrophages (BMMs) (M-BMMs) or GM-CSF–induced BMMs (GM-BMMs) (24). Immortalized macrophage cell lines were established from A-FABP–deficient (A-FABP−/− macrophages) or WT mice (WT macrophages) (22, 27). In brief, bone marrow cells isolated from WT and A-FABP−/− mice were centrifuged through Fico/Lite-LM to remove RBCs. A total of 2 × 106/ml bone marrow cells were cultured in conditioned RPMI 1640 medium containing J2-CRE virus, 5 μg/ml polybrene, 10 ng/ml M-CSF, 10 μg/ml gentamicin, and 10% FBS for overnight. Nonadherent cells were collected and incubated in fresh conditioned medium as shown earlier for 7 d without disturbance. Virus-transformed colonies were isolated for characterization and further analysis. Macrophage cell lines (2 × 105 cells/ml per well) or primary BMMs were lifted and replated (4 × 105 cells/ml per well) in 24-well plates for overnight, then further treated with designated concentrations of saturated or uFAs (100, 200, and 400 μM, respectively) for 18 h for further analysis.

For analyzing macrophage phenotype, macrophage cell lines or M-BMMs were surface stained for 15–30 min at 4°C in 1% BSA PBS containing different Abs (anti-CD11b, clone M1/70; anti-CD11c, clone HL3; anti-F4/80, clone BM8; anti-MHC class II, clone M5/114.15.2; anti-Ly6C, clone HK1.5; anti-CD36, clone HM36). For intracellular Cer staining, macrophages stimulated with saturated or uFAs (400 μM) were fixed and permeabilized with permeabilization buffer (eBioscience) and stained intracellularly with mAb anti-Cer Ab. All samples were acquired on a FACSCalibur flow cytometer, and data analysis was conducted using FlowJo software (Tree Star).

Macrophage cell lines were cultured on poly-d-lysine–coated coverslips (NeuVitro) in a 24-well plate and were treated with sFAs (SA or PA) or uFAs (OA/LA) for a designated period. After fixation and permeabilization, the cells were stained with Cer or BODIPY 493/503 (Invitrogen) as previously described (24). Nuclei were stained with 0.2 μM DAPI (Invitrogen). Confocal slides were analyzed with Nikon A1 laser scanning confocal microscope.

Macrophage cell death was analyzed by flow cytometric staining for 7-aminoactinomycin D (7-AAD) and annexin V (BD Biosciences). Cell viability assay was performed with CellTiter-Glo luminescent viability assay (G7570; Promega).

For measurement of A-FABP and E-FABP protein levels in macrophages, macrophage cell lines or M-BMMs were lysed in buffers with protease inhibitors. Protein concentration was determined by bicinchoninic acid (BCA) assay (Thermo Scientific). Anti-mouse E-FABP and A-FABP Abs (R&D Systems) were used for E-FABP and A-FABP blotting. β-Actin (Cell Signaling) was quantified as a loading control. Image Quant TL system was used for relative protein quantification.

For real-time PCR analysis, RNA was extracted from cells using RNeasy Mini Kit (Qiagen). cDNA synthesis was performed with QuantiTect Reverse Transcription Kit (Qiagen). Quantitative PCR was performed with SYBR Green PCR Master Mix using ABI StepOnePlus Real-Time PCR Systems (Applied Biosystems) to analyze the expression of A-FABP, E-FABP, IL-1α, IL-1β, FA transport protein (FATP) 1, FATP4, TNF-α, CerS2, CerS5, CerS6. Relative mRNA levels were determined using β-actin or HPRT1 as a reference gene.

Control or sFA-treated macrophage cells were pelleted and fixed in 0.1 M phosphate buffer with 2% paraformaldehyde and 3% glutaraldehyde and with 0.1% OsO4, respectively. Samples were dehydrated in an ethanol series and then embedded in plastic resin for sections. Ultrathin sections of 60 nm were cut using a diamond knife (Diatome, Fort Washington, PA). Cell ultrastructural images were examined with transmission electron microscopy at the Research and Innovation Research Core in the University of Louisville.

Duplex small interfering RNAs (siRNAs) targeting the coding region of A-FABP or Cer synthase (CerS) 5 were ordered from Integrated DNA Technologies. To knock down gene expression, WT BMM and macrophages (cell line) were transfected with designated siRNA (20–200 nM) using Oligofectamine (Life Technologies) for 24–48 h before treatments.

Mouse IL-1α, IL-1β, and TNF-α levels in cell culture supernatants or in serum were measured using ELISA kits from BioLegend. Cer species were analyzed using lipid mass spectrophotometry at Metabolomics Core at Mayo Clinic.

Student t test was performed for the comparison of results from different treatments. A p value <0.05 is considered statistically significant.

Because obesity was accompanied by low-grade chronic inflammation and elevated levels of FAs in the circulation, we reasoned that elevated circulating FAs may directly cause cellular damage contributing to chronic inflammation in obesity. Because macrophages were the major immune cells mediating lipid uptake and metabolism in peripheral circulation (28, 29), we generated macrophage cell lines in vitro and cultured them with different concentrations of dietary sFAs or uFAs. Macrophage cell death was monitored by 7-AAD staining using flow cytometry. Interestingly, sFAs (containing PA and SA) induced macrophage cell death in a dose-dependent manner, whereas uFAs (containing OA and LA) exhibited no effects on macrophage survival (Fig. 1A, 1B). Using a luminescent cell viability assay, we consistently observed that sFAs, but not uFAs, significantly decreased macrophage viability (Fig. 1C). To dissect the effect of individual dietary FAs on macrophage cell death, we cultured macrophages with individual FA. As shown in Fig. 1D, both PA and SA potently induced macrophage cell death, whereas OA and LA had no or limited effects on macrophages. When we stained sFA-induced cell death using annexin V, we found that the majority of dying cells were annexin V–negative (Fig. 1E). Transmission electron microscopy showed that dying cells lost their membrane integrity without vesicle formation (Fig. 1F), exhibiting a typical necrotic morphology for sFA-induced macrophage cell death. We also measured the effect of ω-3 FAs on macrophage cell death. Eicosapentaenoic acid appeared to induce macrophage cell death only at very high concentrations (400 μM) (Supplemental Fig. 1). Considering the relatively low levels of ω-3 FAs in the circulation (30), sFAs, but not uFAs, are likely to be the major dietary FAs that induce necrotic macrophage cell death in obesity.

FIGURE 1.

sFAs, but not uFAs, induce macrophage cell death. (A and B) Immortalized macrophages were cultured in the presence or absence of indicated total concentrations of saturated (PA+SA) or uFAs (OA+LA) for 24 h. Flow cytometric analysis of macrophage cell death by 7-AAD staining. The mean percentage and SD of macrophage cell death are shown in (B). (C) Analysis of immortalized macrophage cell viability in response to indicated concentrations of sFAs or uFAs using luminescent viability assay. (D) The mean percentage and SD of immortalized macrophage cell death in response to indicated concentrations of individual FAs. (E) Analyses of sFA (400 μM)–induced macrophage cell death by flow cytometric staining with annexin V and 7-AAD. All data are representative of four independent experiments. Representative electron microscopy images of sFA-induced macrophage cell death are shown in (F). **p < 0.01, ***p < 0.001 compared with the BSA control group.

FIGURE 1.

sFAs, but not uFAs, induce macrophage cell death. (A and B) Immortalized macrophages were cultured in the presence or absence of indicated total concentrations of saturated (PA+SA) or uFAs (OA+LA) for 24 h. Flow cytometric analysis of macrophage cell death by 7-AAD staining. The mean percentage and SD of macrophage cell death are shown in (B). (C) Analysis of immortalized macrophage cell viability in response to indicated concentrations of sFAs or uFAs using luminescent viability assay. (D) The mean percentage and SD of immortalized macrophage cell death in response to indicated concentrations of individual FAs. (E) Analyses of sFA (400 μM)–induced macrophage cell death by flow cytometric staining with annexin V and 7-AAD. All data are representative of four independent experiments. Representative electron microscopy images of sFA-induced macrophage cell death are shown in (F). **p < 0.01, ***p < 0.001 compared with the BSA control group.

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Because PA and OA are the most common FAs in humans, we selected PA and OA as the model of sFA and uFA, respectively, in most of our experiments. Obesity-related cytokines (e.g., TNF-α, IL-1) were shown to promote metabolic inflammation and to exert cytotoxic effects (31, 32). To investigate how sFAs induced macrophage cell death, we first analyzed macrophage cytokine production in response to PA or OA treatment. Neither FA induced apparent TNF-α, IL-1β, or IL-1α production by macrophages (Supplemental Fig. 2A–C), suggesting that these cytokines are not the major effectors mediating PA-induced macrophage cell death. Given that ROS is one of the major mechanisms inducing cell death and can be activated by the sFA palmitate in macrophages (26, 33), we next determined whether ROS was involved in sFA-induced macrophage cell death. Inhibition of ROS generation by specific ROS inhibitors APDC or BHA showed no effects on PA-induced macrophage cell death (Fig. 2A, 2B). In addition, treatment with either PA or OA did not stimulate inducible NO synthase (iNOS) production in macrophages (Supplemental Fig. 2D). Thus, ROS did not seem to be required for sFA-induced macrophage cell death. We also included pan-caspase or specific caspase inhibitors in the cultures and found that caspases were not involved in sFA-induced cell death either (Supplemental Fig. 2E).

FIGURE 2.

Cers are the major mediators of sFA-induced macrophage cell death. (AC) Average percentage of cell death of immortalized macrophages in response to BSA control or 400 μM PA treatment with or without indicated concentrations of ROS inhibitor APDC (A), BHA (B), or triacsin C (2 μM) (C) for 24 h. (D and E) Histogram of intracellular staining of Cers in immortalized macrophages in response to 400 μM PA treatment with or without CerS inhibitor FB1 (10 μM). Mean fluorescent intensities (MFIs) of Cers are shown in (E). (F) Average percentage of cell death of immortalized macrophages in response to BSA control or 400 μM PA treatment with or without indicated concentrations of FB1. Data represent three independent experiments (*p < 0.05, **p < 0.01).

FIGURE 2.

Cers are the major mediators of sFA-induced macrophage cell death. (AC) Average percentage of cell death of immortalized macrophages in response to BSA control or 400 μM PA treatment with or without indicated concentrations of ROS inhibitor APDC (A), BHA (B), or triacsin C (2 μM) (C) for 24 h. (D and E) Histogram of intracellular staining of Cers in immortalized macrophages in response to 400 μM PA treatment with or without CerS inhibitor FB1 (10 μM). Mean fluorescent intensities (MFIs) of Cers are shown in (E). (F) Average percentage of cell death of immortalized macrophages in response to BSA control or 400 μM PA treatment with or without indicated concentrations of FB1. Data represent three independent experiments (*p < 0.05, **p < 0.01).

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Long-chain FAs, either from exogenous or endogenous sources, have to be catalyzed by acyl-CoA synthetase to form acyl-CoAs before they can be used in multiple metabolic pathways (34), so we used triacsin C to inhibit acyl-CoA synthetase activity to determine whether PA-induced cell death can be suppressed. Triacsin C lowered the percentage of PA-induced macrophage cell death from 45 to 20% (Fig. 2C), suggesting that sFAs are activated to produce metabolites that cause cell death. Because PA could be metabolized for synthesis of Cers (35), we showed that PA treatment induced Cer production in macrophages, and PA-induced Cers were able to be inhibited by FB1, a potent inhibitor of CerSs (Fig. 2D, 2E). We further measured PA-induced macrophage cell death in the absence or presence of FB1 (Fig. 2F). Interestingly, FB1 exhibited the similar effects as triacsin C to inhibit PA-induced cell death. Altogether, these data suggest that Cers are the major metabolites mediating sFA-induced macrophage cell death.

Because uFAs were not toxic to macrophages, we speculated that uFAs did not induce Cer synthesis in macrophages. Flow cytometric intracellular staining clearly showed that PA, but not OA or LA, significantly induced Cer formation in macrophages (Fig. 3A, 3B). To investigate why uFAs failed to induce Cers in macrophages, we found that OA, but not PA, was largely stored in the form of lipid droplets in macrophages (Fig. 3C), implying that they are sequestered and unavailable to mediate Cer synthesis. To dissect which species of Cers were critical for PA-induced macrophage cell death, we quantified different Cers of macrophages in the presence or absence of PA treatment. Interestingly, PA increased the generation of all the major species of Cers, including C16-, C18-, C20-, C22-, C24-Cer, in macrophages (Fig. 3D). Considering that C16-Cer was the most abundant species and CerS5 selectively synthesizes C16-Cers (36), we measured the expression of CerSs in macrophages and found that CerS5, but not CerS2 and CerS6, was significantly upregulated in response to PA treatment (Fig. 3E). We further transfected macrophages with CerS5 siRNA to knockdown CerS5 expression (Fig. 3F) and found that PA-induced Cer production (Fig. 3G) and cell death (Fig. 3H) was significantly inhibited when CerS5 was silenced. Taken together, our data indicate that excess sFAs promote Cer synthesis in macrophages, thereby leading to cell death.

FIGURE 3.

sFAs promote Cer production in macrophages. (A and B) Intracellular staining of Cer production in immortalized macrophages treated with 400 μM of PA, OA, or LA, respectively, for 24 h. Mean fluorescent intensity (MFI) of Cer staining is shown in (B). (C) Confocal analysis of lipid droplet formation by BODIPY staining (green) in immortalized macrophages (DAPI staining, blue) treated with BSA control, uFAs (400 μM), or sFAs (400 μM) for 24 h, same as for (A). (D) Measurement of individual Cer species in immortalized macrophages treated with BSA control or PA for 24 h by lipid mass spectrophotometry. (E) Real-time PCR analysis of the expression of CerS2, CerS5, and CerS6 in immortalized macrophages treated with BSA control or PA (400 μM) for 24 h. (F) Real-time PCR analysis of CerS5 expression in immortalized macrophages transfected with scramble RNA control or CerS5-specific siRNA. (G and H) Immortalized macrophages transfected with scramble RNA or CerS5 siRNA were treated with 400 μM of PA for 24 h. PA-induced Cer production (G) was analyzed by intracellular staining, and macrophage cell death (H) was measured by flow cytometric staining with 7-AAD. Experiments were performed a minimum of three times (*p < 0.05, **p < 0.01).

FIGURE 3.

sFAs promote Cer production in macrophages. (A and B) Intracellular staining of Cer production in immortalized macrophages treated with 400 μM of PA, OA, or LA, respectively, for 24 h. Mean fluorescent intensity (MFI) of Cer staining is shown in (B). (C) Confocal analysis of lipid droplet formation by BODIPY staining (green) in immortalized macrophages (DAPI staining, blue) treated with BSA control, uFAs (400 μM), or sFAs (400 μM) for 24 h, same as for (A). (D) Measurement of individual Cer species in immortalized macrophages treated with BSA control or PA for 24 h by lipid mass spectrophotometry. (E) Real-time PCR analysis of the expression of CerS2, CerS5, and CerS6 in immortalized macrophages treated with BSA control or PA (400 μM) for 24 h. (F) Real-time PCR analysis of CerS5 expression in immortalized macrophages transfected with scramble RNA control or CerS5-specific siRNA. (G and H) Immortalized macrophages transfected with scramble RNA or CerS5 siRNA were treated with 400 μM of PA for 24 h. PA-induced Cer production (G) was analyzed by intracellular staining, and macrophage cell death (H) was measured by flow cytometric staining with 7-AAD. Experiments were performed a minimum of three times (*p < 0.05, **p < 0.01).

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To further investigate the mechanisms by which excess sFAs promote Cer synthesis, we focused on the family of FABPs because they are central in regulating FA transport and coordinating lipid responses inside cells (37, 38). Because macrophages are notably heterogeneous and our previous studies demonstrated that the FABP expression pattern was tightly regulated in different subsets of macrophages (24), we first determined the phenotype and FABP expression pattern of the macrophage cell line we generated. As shown in Fig. 4A and 4B, the macrophage cell line exhibited a CD36+ CD11b+ Ly6cF4/80highCD11c phenotype with high expression of both A-FABP and E-FABP, which were different from GM-BMMs with a CD36CD11b+ Ly6cF4/80lowCD11c+ phenotype and predominant E-FABP expression (24). These data suggest an important role of A-FABP in this CD36+ phenotype of macrophages. Importantly, when PA and OA were added in the culture, respectively, only PA treatment significantly increased the expression of A-FABP (Fig. 4C). Western blotting further confirmed the upregulation of A-FABP expression by PA at the protein level (Fig. 4D), implying a critical role of A-FABP in mediating sFA-induced lipid responses in this phenotype of macrophages.

FIGURE 4.

sFAs upregulate A-FABP expression in macrophages. (A) Flow cytometric analysis of the phenotype of immortalized macrophages by staining with anti-CD11b, anti-F4/80, anti-CD36, anti-MHCII, anti-CD11c, and anti-Ly6C. Dotted lines represent isotype control; solid lines represent indicated Ab staining. (B) Real-time PCR analysis of expression of FABP family members in immortalized macrophages. (C and D) Analysis of A-FABP and E-FABP expression by real-time PCR (C) and Western blotting (D) in macrophages treated with BSA or PA for 15 h. Data are representative of three independent experiments (*p < 0.05).

FIGURE 4.

sFAs upregulate A-FABP expression in macrophages. (A) Flow cytometric analysis of the phenotype of immortalized macrophages by staining with anti-CD11b, anti-F4/80, anti-CD36, anti-MHCII, anti-CD11c, and anti-Ly6C. Dotted lines represent isotype control; solid lines represent indicated Ab staining. (B) Real-time PCR analysis of expression of FABP family members in immortalized macrophages. (C and D) Analysis of A-FABP and E-FABP expression by real-time PCR (C) and Western blotting (D) in macrophages treated with BSA or PA for 15 h. Data are representative of three independent experiments (*p < 0.05).

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To determine whether A-FABP upregulation promoted sFA-induced Cer production in macrophages, we first transfected macrophages with A-FABP siRNA to knock down A-FABP expression (Fig. 5A) and then measured PA-induced Cer production in these macrophages. Flow cytometric intracellular staining showed that PA-induced Cer production was significantly reduced when A-FABP was knocked down by siRNA in macrophages (Fig. 5B, 5C). Next, we used our unique A-FABP–deficient macrophages to confirm the earlier observations (Fig. 5D). As shown by intracellular staining of Cers, A-FABP–deficient macrophages exhibited a more dramatic reduction in PA- or SA-induced Cer production as compared with A-FABP–sufficient WT macrophages. OA did not induce Cer production in both types of macrophages (Fig. 5E). Finally, we measured PA-induced production of different Cer species in WT and A-FABP–deficient macrophages using lipid mass spectrometry. Except C14 Cer (Fig. 5F), production of long-chain species of Cers, including C16, C18, C20, C22, C24, S1P, and sphinganine (Fig. 5G–M), was significantly inhibited in the absence of A-FABP in macrophages. Integration of all these results indicates that A-FABP is essential in promoting sFA-induced Cer production in macrophages.

FIGURE 5.

PA-induced Cer production in macrophages is regulated by A-FABP expression. (A) Real-time PCR analysis of A-FABP knockdown by specific siRNA for A-FABP in the WT macrophage cell line. (B and C) Intracellular staining of PA-induced Cer production in WT macrophage cell line transfected with scramble or A-FABP–specific siRNA. Mean fluorescent intensity (MFI) of Cer was shown in (C). (D) Analysis of A-FABP expression in WT and A-FABP−/− macrophage cell lines by real-time RT-PCR. (E) Analysis of MFI of Cer production in WT and A-FABP−/− macrophage cell lines after treatment with PA, SA, or OA (400 μM) for 18 h by intracellular flow cytometric staining. (FM) Measurement of levels of C14-Cer (F), C16-Cer (G), C18-Cer (H), C20-Cer (I), C22-Cer (J), S1P (K), sphinganine (L), and C24-1 Cer (M) in WT and A-FABP−/− macrophage cell lines treated with PA for 18 h by mass spectrometry. Data are representative of three independent experiments (*p < 0.05, **p < 0.01).

FIGURE 5.

PA-induced Cer production in macrophages is regulated by A-FABP expression. (A) Real-time PCR analysis of A-FABP knockdown by specific siRNA for A-FABP in the WT macrophage cell line. (B and C) Intracellular staining of PA-induced Cer production in WT macrophage cell line transfected with scramble or A-FABP–specific siRNA. Mean fluorescent intensity (MFI) of Cer was shown in (C). (D) Analysis of A-FABP expression in WT and A-FABP−/− macrophage cell lines by real-time RT-PCR. (E) Analysis of MFI of Cer production in WT and A-FABP−/− macrophage cell lines after treatment with PA, SA, or OA (400 μM) for 18 h by intracellular flow cytometric staining. (FM) Measurement of levels of C14-Cer (F), C16-Cer (G), C18-Cer (H), C20-Cer (I), C22-Cer (J), S1P (K), sphinganine (L), and C24-1 Cer (M) in WT and A-FABP−/− macrophage cell lines treated with PA for 18 h by mass spectrometry. Data are representative of three independent experiments (*p < 0.05, **p < 0.01).

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To further investigate the role of A-FABP in sFA-mediated macrophage cell death, we treated macrophages with different types of dietary FAs and measured their cell death. Strikingly, deficiency of A-FABP significantly suppressed both PA- and SA-induced macrophage cell death. Consistent with the earlier Cer data, treatment with OA did not induce macrophage cell death regardless of A-FABP expression (Fig. 6A). We also knocked down A-FABP expression with A-FABP siRNA in WT macrophages and confirmed that A-FABP deficiency indeed inhibited PA-induced macrophage cell death (Supplemental Fig. 3A). Moreover, dual staining for Cer+ and 7-AAD+ dead cells with flow cytometry clearly showed that A-FABP deficiency specifically inhibited sFA-induced Cer+ dead cells (Q2 population), but not Cer dead cells (Q1 population) (Fig. 6B), indicating a specific role of A-FABP in promoting Cer-mediated cell death. To determine how A-FABP promoted sFA-induced cell death, we found that A-FABP deficiency neither impacted the expression of major FATPs (Supplemental Fig. 3B, 3C) nor affected FA uptake by macrophages (Fig. 6C). However, when we compared lipid droplet formation after FA uptake between WT and A-FABP−/− macrophages, we found that more lipids were stored in the form of lipid droplets in the A-FABP−/− macrophages (Fig. 6D, 6E). These data suggest that A-FABP deficiency may not impact lipid uptake, but rather restrict lipid utilization, thereby lowering Cer synthesis in macrophages.

FIGURE 6.

A-FABP deficiency protects macrophages against sFA-induced Cer-mediated cell death. (A) Measurement of different types of FA–induced cell death (400 μM for each FA) using WT and A-FABP−/− macrophage cell lines. (B) Dual staining of 7-AAD and surface Cer in WT and A-FABP−/− macrophage cell lines after 18-h individual FA treatment (PA, SA, OA, 400 μM, BSA as control). (C) WT and A-FABP−/− macrophage cell lines (0.2 × 106/ml per well) were cultured with designated concentrations of BODIPY-FL-C16 for 30 min; then cells were washed and harvested for flow cytometric analysis. (D and E) WT and A-FABP−/− macrophage cell lines (0.2 × 106/ml per well) were cultured for 16 h with 100 μM PA, SA, or OA on round coverslips in a 24-well plate. Cells were fixed and stained with BODIPY (green) and DAPI (blue) by confocal analysis (D). The fluorescence intensity of 12 typical fluorescent sites from each image was quantified with the background subtracted in Nikon NIS elements image software (E). Experiments were performed a minimum of three times (**p < 0.01, ***p < 0.0001 as compared with the WT control).

FIGURE 6.

A-FABP deficiency protects macrophages against sFA-induced Cer-mediated cell death. (A) Measurement of different types of FA–induced cell death (400 μM for each FA) using WT and A-FABP−/− macrophage cell lines. (B) Dual staining of 7-AAD and surface Cer in WT and A-FABP−/− macrophage cell lines after 18-h individual FA treatment (PA, SA, OA, 400 μM, BSA as control). (C) WT and A-FABP−/− macrophage cell lines (0.2 × 106/ml per well) were cultured with designated concentrations of BODIPY-FL-C16 for 30 min; then cells were washed and harvested for flow cytometric analysis. (D and E) WT and A-FABP−/− macrophage cell lines (0.2 × 106/ml per well) were cultured for 16 h with 100 μM PA, SA, or OA on round coverslips in a 24-well plate. Cells were fixed and stained with BODIPY (green) and DAPI (blue) by confocal analysis (D). The fluorescence intensity of 12 typical fluorescent sites from each image was quantified with the background subtracted in Nikon NIS elements image software (E). Experiments were performed a minimum of three times (**p < 0.01, ***p < 0.0001 as compared with the WT control).

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After demonstrating an essential role of A-FABP in mediating sFA-induced cell death in macrophage cell lines, we further validated these findings using primary BMMs. As compared with GM-BMMs, M-BMMs expressed high levels of CD36, but low levels of CD11c, which exhibited similar phenotype (CD36+CD11b+F4/80+ CD11c) as the macrophage cell lines (Fig. 7A). More importantly, M-BMMs had higher levels of A-FABP than GM-BMMs at both RNA levels and protein levels (Fig. 7B, 7C). We thus used these A-FABP+CD36+ M-BMMs from WT and A-FABP−/− mice to evaluate the role of A-FABP in our studies. Although PA-induced cell death was much lower in primary M-BMMs as compared with macrophage cell lines, A-FABP deficiency in BMMs reduced cell death induced by PA by ∼50% (Fig. 7D). Moreover, in line with the results obtained from macrophage cell lines, A-FABP deficiency also significantly reduced SA-induced cell death of M-BMMs (Fig. 7E). Interestingly, when WT and A-FABP−/− mice were fed a high-fat diet to induce obesity, A-FABP deficiency did not impact mouse weight increase (Supplementary Fig. 4A), nor the percentage and phenotype of monocytes in the bone marrow (Supplementary Fig. 4B, 4C). However, the percentage of peripheral CD36+ monocytes in obese A-FABP−/− mice was significantly higher than those in obese WT mice (Supplementary Fig. 4D, 4E). Although in vivo animal studies were more complicated than in vitro cell-culture experiments, the observed in vivo data provided evidence to support our in vitro observations that A-FABP deficiency protects monocytes/macrophages from dietary FA–induced cell death, thus contributing to the elevated A-FABP+ CD36+ monocyte subsets in obese A-FABP−/− mice.

FIGURE 7.

A-FABP deficiency reduced sFA-induced cell death in primary BMMs. (A) Flow cytometric analysis of the phenotype of M-BMMs and GM-BMMs with indicated mAbs. (B and C) Comparison of A-FABP expression between M-BMMs and GM-BMMs by real-time RT-PCR (B) and Western blotting (C). (D and E) Primary M-BMMs from WT and A-FABP−/− mice were plated in a 24-well plate (0.4 × 106/ml per well) and treated with PA (D) or SA (E) (400 μM) for 24 h. Cell death was analyzed with flow staining for 7-AAD. All experiments were performed at least three times (*p < 0.05, **p < 0.01).

FIGURE 7.

A-FABP deficiency reduced sFA-induced cell death in primary BMMs. (A) Flow cytometric analysis of the phenotype of M-BMMs and GM-BMMs with indicated mAbs. (B and C) Comparison of A-FABP expression between M-BMMs and GM-BMMs by real-time RT-PCR (B) and Western blotting (C). (D and E) Primary M-BMMs from WT and A-FABP−/− mice were plated in a 24-well plate (0.4 × 106/ml per well) and treated with PA (D) or SA (E) (400 μM) for 24 h. Cell death was analyzed with flow staining for 7-AAD. All experiments were performed at least three times (*p < 0.05, **p < 0.01).

Close modal

Obesity is associated with low-grade chronic inflammation without bacterial or viral infection, but the triggers and molecular mechanisms that lead to obesity-associated metabolic inflammation remain to be further explored. Although recent studies demonstrated palmitate triggered thioglycollate-elicited macrophage death under the stimulation of Gram-negative bacteria-derived LPS (39, 40), it did not explain the sterile inflammation associated with obesity, and it also raised concerns regarding physiology and activated status of these thioglycollate-activated macrophages (41). In this article, we report that when various dietary FAs were uptaken by stable macrophage cell lines or primary BMMs, only sFAs (e.g., PA, SA) were metabolized to produce Cers to induce macrophage cell death. Most importantly, we identified A-FABP as a new molecular sensor in mediating excess sFA-induced Cer production and in promoting macrophage cell death, thus contributing to the sterile chronic inflammation in obesity.

In this study we addressed several critical questions regarding obesity-associated inflammation. First, many endogenous and exogenous triggers have been shown to induce sterile inflammation during obesity (31), but whether upregulated levels of FAs can serve as sterile stimuli in obesity-associated inflammation has been unclear. To this end, we cultured macrophages in the presence of different types of dietary FAs in vitro and observed their responses. We observed that excess sFA (e.g., PA and SA), but not uFAs (e.g., OA and LA), induced significant cell death after overnight culture. These results are consistent with other studies using myocytes or hepatocytes (16, 42). In determining the specific effects of sFAs in inducing macrophage cell death, we found that sFAs are metabolized to produce cytotoxic Cers in macrophages, which can be inhibited either by silencing CerS5 or by FB1, a specific CerS inhibitor. In contrast, uFAs are sequestered in lipid droplets averting away Cer pathways. Thus, our data reveal sFAs as new sterile stimuli inducing macrophage cell death. Notably, cytotoxic effects of sFAs have been shown in various types of cells, including heart tissue–derived H9C2 cells and ovary cells, through generation of ROS (43, 44). However, ROS did not appear to be a major mechanism in mediating sFA-induced cell death in macrophages in that: 1) sFA-induced macrophage cell death started early 12 h before iNOS was majorly produced; 2) both nontoxic OA and toxic PA induced equivalent amounts of iNOS production in macrophages; and 3) ROS-specific inhibitors, including APDC and BHA, exhibited only marginal effects on PA-induced macrophage death, which was consistent with previous studies showing Cer-induced, ROS-independent necrosis in Jurkat and U937 cells (45, 46). Instead, in response to environmental stimuli, macrophages produced large amounts of ROS for maintaining immune surveillance and tissue homeostasis (47, 48). Moreover, ROS produced by macrophages can even promote their survival and differentiation (47, 48). Thus, compared with nonphagocytic cells, phagocytic macrophages exhibit unique mechanisms in response to excess sFAs in the setting of obesity.

Second, although macrophages have been established as major phagocytic cells in clearing excess lipids during obesity, they are notoriously heterogeneous with different phenotypes and functions in vivo. This leads to the question: do different subsets of macrophages respond equally to individual dietary FAs? Using bone marrow cells, we can differentiate different phenotypes of macrophages with M-CSF or GM-CSF. M-BMMs exhibit CD11b+F4/80+Ly6CCD11cCD36+ phenotype (M2-like), whereas GM-BMMs are CD11b+F4/80+Ly6CCD11c+CD36 (M1-like). In peripheral blood, monocytes can also be generally divided into F4/80+CD36+ and F4/80+CD36 subsets. Interestingly, we found that sFAs (e.g., SA) induced significant cell death of macrophage cell lines and M-BMMs, which highly express CD36. In sharp contrast, they exerted minimal cytotoxic effects on either CD36 GM-BMMs or CD36 peripheral monocytes. These observations were substantiated by previous studies showing that CD36 expression in macrophages facilitate lipid uptake and metabolism in atherosclerosis (49). Thus, our results suggest CD36+ M2-like, but not CD36 M1-like, monocytes/macrophages are the major subsets prone to cell death in the periphery and adipose tissues in obesity, supporting the idea that individual monocyte/macrophage subsets have their unique features in mediating FA metabolism.

Third, what kinds of molecular sensors in individual macrophage subsets may determine their unique responsiveness to FAs? Given the central role of FABPs in coordinating FA transportation and responses, we analyzed the profile of the FABP family in CD36+ M-BMMs and CD36 GM-BMMs, and found that A-FABP expression compared with other FABPs was specifically upregulated in M-GMMs, suggesting a unique role of A-FABP in meditating lipid metabolism in these cells (50). In line with our observations, A-FABP has been shown to be positively associated with CD36 expression by activated PPARγ in macrophages (5153). Our previous studies have shown that E-FABP, but not A-FABP, is highly expressed CD11c+ CD36 macrophages in different settings of diseases (10, 24). Thus, the distribution pattern of FABP expression may directly determine FA metabolic pathways and responses in different subsets of macrophages. Considering that A-FABP is more strictly expressed in lipid-laden adipocytes and CD36+ macrophage subsets, we reason that expression of A-FABP may function as molecular sensors detecting excess FAs in these cells. In contrast, E-FABP is widely expressed in different types of lipid-scarce immune populations, including T cells (19). Expression of E-FABP may sense and partition FAs for essential energy production, maintaining fundamental cellular function and activity. Moreover, high expression of E-FABP in CD11c+ CD36 macrophages also facilitates FA–induced lipid droplet formation (24), and thus may reduce FA-mediated cytotoxic metabolic pathways. This may explain why CD36 macrophages are more tolerant than CD36+ macrophages in response to sFA-induced cell death.

Last, to determine whether A-FABP is essential in mediating sFA-induced Cer production and macrophage cell death, we either knocked down A-FABP expression with RNA silencing or abrogated A-FABP expression using A-FABP–deficient macrophage cell lines. Both strategies demonstrated that A-FABP expression is indeed critical in facilitating sFA-induced Cer production and cytotoxicity. Further analyses indicate that A-FABP deficiency affects neither FA uptake nor expression of CerSs in macrophages. However, internalized FAs in A-FABP–deficient macrophages were more stored in the lipid droplets than these in A-FABP–sufficient macrophages, which are consistent with the observation of high levels of FAs in A-FABP–deficient macrophages (54). Using bone marrow–derived primary macrophages (M-BMMs), although exhibiting relative resistance to PA treatment as compared with SA treatment, we demonstrated that A-FABP deficiency significantly inhibited both PA- and SA-induced cell death in primary macrophages. Finally, we observed that obese WT mice exhibited less percentage of peripheral CD36+ monocytes than obese A-FABP−/− mice, which supports our in vitro results that A-FABP expression promotes CD36+ macrophage cell death. Of note, the critical role of A-FABP in promoting sFA-induced monocyte/macrophage cell death was not observed when we analyzed monocyte populations using bone marrow cells from WT and A-FABP−/− mice, which could be due to the fact that A-FABP is not or is very lowly expressed in immature bone marrow cells. We also noticed that neither inhibition of Cer synthesis nor deficiency of A-FABP expression was able to completely inhibit sFA-induced macrophage cell death, suggesting that other signaling pathways are also involved in sFA-induced cell death. Nonetheless, the current studies enhance our understanding why inhibition of A-FABP ameliorates obesity-associated diseases including atherosclerosis, diabetes, and other metabolic syndromes (55, 56), and provide mechanistic evidence by establishing A-FABP as a new molecular sensor for sFA-induced Cer synthesis and cell death of macrophages.

In summary, our current studies establish dietary FAs, especially sFAs, as new triggers of inducing cell death through promotion of cytotoxic Cer synthesis in macrophages. Most importantly, our results demonstrate that inhibition of A-FABP expression in CD36+ macrophages suppresses sFA-induced Cer production and cell death, thereby identifying A-FABP as a new molecular sensor in sFA-mediated responses in specific subsets of macrophages. Thus, it demonstrates a novel mechanism by which A-FABP promotes Cer production by excess dietary FAs in macrophages and contributes to obesity-associated chronic inflammation.

We thank Dr. Xuan-Mai T. Person and Dr. Michael D. Jensen for Cer species analysis with lipid mass spectrometry. We thank Dr. Brian Watterberg for critical reading and helpful suggestions for the manuscript. We also thank Dr. Martha Bickford and Arkadiusz Slusarczyk for help with the electron microscopy.

This work was supported by National Institutes of Health R01 Grants CA18098601A1 and CA17767901A1, National Institute of Diabetes and Digestive and Kidney Diseases Grant U24DK100469, and the National Center for Advancing Translational Sciences (UL1TR000135).

The online version of this article contains supplemental material.

Abbreviations used in this article:

7-AAD

7-aminoactinomycin D

A-FABP

adipose FABP

APDC

4-amino-2,4-pyrrolidine-dicarboxylic acid

BHA

butylated hydroxyanisole

BMM

bone marrow–derived macrophage

Cer

ceramide

CerS

Cer synthase

E-FABP

epidermal FABP

FA

fatty acid

FABP

FA binding protein

FATP

FA transport protein

FB1

fumonisin B1

GM-BMM

GM-CSF–induced BMM

iNOS

inducible NO synthase

LA

linoleic acid

M-BMM

M-CSF–induced bone marrow–derived macrophage

OA

oleic acid

PA

palmitic acid

ROS

reactive oxygen species

SA

stearic acid

sFA

saturated FA

siRNA

small interfering RNA

uFA

unsaturated FA

WT

wild type.

1
Gregor
M. F.
,
Hotamisligil
G. S.
.
2011
.
Inflammatory mechanisms in obesity.
Annu. Rev. Immunol.
29
:
415
445
.
2
Khandekar
M. J.
,
Cohen
P.
,
Spiegelman
B. M.
.
2011
.
Molecular mechanisms of cancer development in obesity.
Nat. Rev. Cancer
11
:
886
895
.
3
Amar
S.
,
Zhou
Q.
,
Shaik-Dasthagirisaheb
Y.
,
Leeman
S.
.
2007
.
Diet-induced obesity in mice causes changes in immune responses and bone loss manifested by bacterial challenge.
Proc. Natl. Acad. Sci. USA
104
:
20466
20471
.
4
Zhou
Q.
,
Leeman
S. E.
,
Amar
S.
.
2011
.
Signaling mechanisms in the restoration of impaired immune function due to diet-induced obesity.
Proc. Natl. Acad. Sci. USA
108
:
2867
2872
.
5
de Heredia
F. P.
,
Gómez-Martínez
S.
,
Marcos
A.
.
2012
.
Obesity, inflammation and the immune system.
Proc. Nutr. Soc.
71
:
332
338
.
6
Weisberg
S. P.
,
McCann
D.
,
Desai
M.
,
Rosenbaum
M.
,
Leibel
R. L.
,
Ferrante
A. W.
 Jr.
2003
.
Obesity is associated with macrophage accumulation in adipose tissue.
J. Clin. Invest.
112
:
1796
1808
.
7
Subramanian
V.
,
Ferrante
A. W.
 Jr.
2009
.
Obesity, inflammation, and macrophages.
Nestle Nutr. Workshop Ser. Pediatr. Program.
63
:
151
159
.discussion 159–162, 259–268.
8
Moore
K. J.
,
Sheedy
F. J.
,
Fisher
E. A.
.
2013
.
Macrophages in atherosclerosis: a dynamic balance.
Nat. Rev. Immunol.
13
:
709
721
.
9
Olefsky
J. M.
,
Glass
C. K.
.
2010
.
Macrophages, inflammation, and insulin resistance.
Annu. Rev. Physiol.
72
:
219
246
.
10
Zhang
Y.
,
Li
Q.
,
Rao
E.
,
Sun
Y.
,
Grossmann
M. E.
,
Morris
R. J.
,
Cleary
M. P.
,
Li
B.
.
2015
.
Epidermal fatty acid binding protein promotes skin inflammation induced by high-fat diet.
Immunity
42
:
953
964
.
11
Boden
G.
2011
.
Obesity, insulin resistance and free fatty acids.
Curr. Opin. Endocrinol. Diabetes Obes.
18
:
139
143
.
12
Frayn
K. N.
,
Williams
C. M.
,
Arner
P.
.
1996
.
Are increased plasma non-esterified fatty acid concentrations a risk marker for coronary heart disease and other chronic diseases?
Clin. Sci.
90
:
243
253
.
13
Couillard
C.
,
Bergeron
N.
,
Prud’homme
D.
,
Bergeron
J.
,
Tremblay
A.
,
Bouchard
C.
,
Mauriège
P.
,
Després
J. P.
.
1998
.
Postprandial triglyceride response in visceral obesity in men.
Diabetes
47
:
953
960
.
14
Mei
S.
,
Ni
H. M.
,
Manley
S.
,
Bockus
A.
,
Kassel
K. M.
,
Luyendyk
J. P.
,
Copple
B. L.
,
Ding
W. X.
.
2011
.
Differential roles of unsaturated and saturated fatty acids on autophagy and apoptosis in hepatocytes.
J. Pharmacol. Exp. Ther.
339
:
487
498
.
15
Shimabukuro
M.
,
Zhou
Y. T.
,
Levi
M.
,
Unger
R. H.
.
1998
.
Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes.
Proc. Natl. Acad. Sci. USA
95
:
2498
2502
.
16
de Vries
J. E.
,
Vork
M. M.
,
Roemen
T. H.
,
de Jong
Y. F.
,
Cleutjens
J. P.
,
van der Vusse
G. J.
,
van Bilsen
M.
.
1997
.
Saturated but not mono-unsaturated fatty acids induce apoptotic cell death in neonatal rat ventricular myocytes.
J. Lipid Res.
38
:
1384
1394
.
17
Furuhashi
M.
,
Hotamisligil
G. S.
.
2008
.
Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets.
Nat. Rev. Drug Discov.
7
:
489
503
.
18
Chmurzyńska
A.
2006
.
The multigene family of fatty acid-binding proteins (FABPs): function, structure and polymorphism.
J. Appl. Genet.
47
:
39
48
.
19
Li
B.
,
Reynolds
J. M.
,
Stout
R. D.
,
Bernlohr
D. A.
,
Suttles
J.
.
2009
.
Regulation of Th17 differentiation by epidermal fatty acid-binding protein.
J. Immunol.
182
:
7625
7633
.
20
Rolph
M. S.
,
Young
T. R.
,
Shum
B. O.
,
Gorgun
C. Z.
,
Schmitz-Peiffer
C.
,
Ramshaw
I. A.
,
Hotamisligil
G. S.
,
Mackay
C. R.
.
2006
.
Regulation of dendritic cell function and T cell priming by the fatty acid-binding protein AP2.
J. Immunol.
177
:
7794
7801
.
21
Zhang
Y.
,
Li
B.
.
2014
.
E-FABP: regulator of immune function.
Oncoscience
1
:
398
399
.
22
Makowski
L.
,
Brittingham
K. C.
,
Reynolds
J. M.
,
Suttles
J.
,
Hotamisligil
G. S.
.
2005
.
The fatty acid-binding protein, aP2, coordinates macrophage cholesterol trafficking and inflammatory activity. Macrophage expression of aP2 impacts peroxisome proliferator-activated receptor gamma and IkappaB kinase activities.
J. Biol. Chem.
280
:
12888
12895
.
23
Reynolds
J. M.
,
Liu
Q.
,
Brittingham
K. C.
,
Liu
Y.
,
Gruenthal
M.
,
Gorgun
C. Z.
,
Hotamisligil
G. S.
,
Stout
R. D.
,
Suttles
J.
.
2007
.
Deficiency of fatty acid-binding proteins in mice confers protection from development of experimental autoimmune encephalomyelitis.
J. Immunol.
179
:
313
321
.
24
Zhang
Y.
,
Sun
Y.
,
Rao
E.
,
Yan
F.
,
Li
Q.
,
Zhang
Y.
,
Silverstein
K. A.
,
Liu
S.
,
Sauter
E.
,
Cleary
M. P.
,
Li
B.
.
2014
.
Fatty acid-binding protein E-FABP restricts tumor growth by promoting IFN-β responses in tumor-associated macrophages.
Cancer Res.
74
:
2986
2998
.
25
Freigang
S.
,
Ampenberger
F.
,
Weiss
A.
,
Kanneganti
T. D.
,
Iwakura
Y.
,
Hersberger
M.
,
Kopf
M.
.
2013
.
Fatty acid-induced mitochondrial uncoupling elicits inflammasome-independent IL-1α and sterile vascular inflammation in atherosclerosis.
Nat. Immunol.
14
:
1045
1053
.
26
Wen
H.
,
Gris
D.
,
Lei
Y.
,
Jha
S.
,
Zhang
L.
,
Huang
M. T.
,
Brickey
W. J.
,
Ting
J. P.
.
2011
.
Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling.
Nat. Immunol.
12
:
408
415
.
27
Clemons-Miller
A. R.
,
Cox
G. W.
,
Suttles
J.
,
Stout
R. D.
.
2000
.
LPS stimulation of TNF-receptor deficient macrophages: a differential role for TNF-alpha autocrine signaling in the induction of cytokine and nitric oxide production.
Immunobiology
202
:
477
492
.
28
McLaren
J. E.
,
Michael
D. R.
,
Ashlin
T. G.
,
Ramji
D. P.
.
2011
.
Cytokines, macrophage lipid metabolism and foam cells: implications for cardiovascular disease therapy.
Prog. Lipid Res.
50
:
331
347
.
29
Shibata
N.
,
Glass
C. K.
.
2009
.
Regulation of macrophage function in inflammation and atherosclerosis.
J. Lipid Res.
50
(
Suppl.
):
S277
S281
.
30
Sekikawa
A.
,
Curb
J. D.
,
Ueshima
H.
,
El-Saed
A.
,
Kadowaki
T.
,
Abbott
R. D.
,
Evans
R. W.
,
Rodriguez
B. L.
,
Okamura
T.
,
Sutton-Tyrrell
K.
, et al
ERA JUMP (Electron-Beam Tomography, Risk Factor Assessment Among Japanese and U.S. Men in the Post-World War II Birth Cohort) Study Group
.
2008
.
Marine-derived n-3 fatty acids and atherosclerosis in Japanese, Japanese-American, and white men: a cross-sectional study.
J. Am. Coll. Cardiol.
52
:
417
424
.
31
Chen
G. Y.
,
Nuñez
G.
.
2010
.
Sterile inflammation: sensing and reacting to damage.
Nat. Rev. Immunol.
10
:
826
837
.
32
Kim
K. A.
,
Lee
M. S.
.
2009
.
Recent progress in research on beta-cell apoptosis by cytokines.
Front. Biosci. (Landmark Ed.)
14
:
657
664
.
33
Orrenius
S.
2007
.
Reactive oxygen species in mitochondria-mediated cell death.
Drug Metab. Rev.
39
:
443
455
.
34
Mashek
D. G.
,
Li
L. O.
,
Coleman
R. A.
.
2007
.
Long-chain acyl-CoA synthetases and fatty acid channeling.
Future Lipidol.
2
:
465
476
.
35
Hannun
Y. A.
,
Obeid
L. M.
.
2008
.
Principles of bioactive lipid signalling: lessons from sphingolipids.
Nat. Rev. Mol. Cell Biol.
9
:
139
150
.
36
Lahiri
S.
,
Futerman
A. H.
.
2005
.
LASS5 is a bona fide dihydroceramide synthase that selectively utilizes palmitoyl-CoA as acyl donor.
J. Biol. Chem.
280
:
33735
33738
.
37
Smathers
R. L.
,
Petersen
D. R.
.
2011
.
The human fatty acid-binding protein family: evolutionary divergences and functions.
Hum. Genomics
5
:
170
191
.
38
Storch
J.
,
Corsico
B.
.
2008
.
The emerging functions and mechanisms of mammalian fatty acid-binding proteins.
Annu. Rev. Nutr.
28
:
73
95
.
39
Schilling
J. D.
,
Machkovech
H. M.
,
He
L.
,
Diwan
A.
,
Schaffer
J. E.
.
2013
.
TLR4 activation under lipotoxic conditions leads to synergistic macrophage cell death through a TRIF-dependent pathway.
J. Immunol.
190
:
1285
1296
.
40
Schilling
J. D.
,
Machkovech
H. M.
,
He
L.
,
Sidhu
R.
,
Fujiwara
H.
,
Weber
K.
,
Ory
D. S.
,
Schaffer
J. E.
.
2013
.
Palmitate and lipopolysaccharide trigger synergistic ceramide production in primary macrophages.
J. Biol. Chem.
288
:
2923
2932
.
41
Zhang
X.
,
Goncalves
R.
,
Mosser
D. M.
.
2008
.
The isolation and characterization of murine macrophages.
Curr. Protoc. Immunol.
Chapter 14
:
Unit 14.1
.
42
Ricchi
M.
,
Odoardi
M. R.
,
Carulli
L.
,
Anzivino
C.
,
Ballestri
S.
,
Pinetti
A.
,
Fantoni
L. I.
,
Marra
F.
,
Bertolotti
M.
,
Banni
S.
, et al
.
2009
.
Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes.
J. Gastroenterol. Hepatol.
24
:
830
840
.
43
Listenberger
L. L.
,
Ory
D. S.
,
Schaffer
J. E.
.
2001
.
Palmitate-induced apoptosis can occur through a ceramide-independent pathway.
J. Biol. Chem.
276
:
14890
14895
.
44
Wei
C. D.
,
Li
Y.
,
Zheng
H. Y.
,
Tong
Y. Q.
,
Dai
W.
.
2013
.
Palmitate induces H9c2 cell apoptosis by increasing reactive oxygen species generation and activation of the ERK1/2 signaling pathway.
Mol. Med. Rep.
7
:
855
861
.
45
Festjens
N.
,
Vanden Berghe
T.
,
Vandenabeele
P.
.
2006
.
Necrosis, a well-orchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response.
Biochim. Biophys. Acta
1757
:
1371
1387
.
46
Thon
L.
,
Möhlig
H.
,
Mathieu
S.
,
Lange
A.
,
Bulanova
E.
,
Winoto-Morbach
S.
,
Schütze
S.
,
Bulfone-Paus
S.
,
Adam
D.
.
2005
.
Ceramide mediates caspase-independent programmed cell death.
FASEB J.
19
:
1945
1956
.
47
Forman
H. J.
,
Torres
M.
.
2001
.
Redox signaling in macrophages.
Mol. Aspects Med.
22
:
189
216
.
48
Zhang
Y.
,
Choksi
S.
,
Chen
K.
,
Pobezinskaya
Y.
,
Linnoila
I.
,
Liu
Z. G.
.
2013
.
ROS play a critical role in the differentiation of alternatively activated macrophages and the occurrence of tumor-associated macrophages.
Cell Res.
23
:
898
914
.
49
Collot-Teixeira
S.
,
Martin
J.
,
McDermott-Roe
C.
,
Poston
R.
,
McGregor
J. L.
.
2007
.
CD36 and macrophages in atherosclerosis.
Cardiovasc. Res.
75
:
468
477
.
50
Makowski
L.
,
Boord
J. B.
,
Maeda
K.
,
Babaev
V. R.
,
Uysal
K. T.
,
Morgan
M. A.
,
Parker
R. A.
,
Suttles
J.
,
Fazio
S.
,
Hotamisligil
G. S.
,
Linton
M. F.
.
2001
.
Lack of macrophage fatty-acid-binding protein aP2 protects mice deficient in apolipoprotein E against atherosclerosis.
Nat. Med.
7
:
699
705
.
51
Sarov-Blat
L.
,
Kiss
R. S.
,
Haidar
B.
,
Kavaslar
N.
,
Jaye
M.
,
Bertiaux
M.
,
Steplewski
K.
,
Hurle
M. R.
,
Sprecher
D.
,
McPherson
R.
,
Marcel
Y. L.
.
2007
.
Predominance of a proinflammatory phenotype in monocyte-derived macrophages from subjects with low plasma HDL-cholesterol.
Arterioscler. Thromb. Vasc. Biol.
27
:
1115
1122
.
52
Chawla
A.
,
Barak
Y.
,
Nagy
L.
,
Liao
D.
,
Tontonoz
P.
,
Evans
R. M.
.
2001
.
PPAR-gamma dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation.
Nat. Med.
7
:
48
52
.
53
Fu
Y.
,
Luo
N.
,
Lopes-Virella
M. F.
,
Garvey
W. T.
.
2002
.
The adipocyte lipid binding protein (ALBP/aP2) gene facilitates foam cell formation in human THP-1 macrophages.
Atherosclerosis
165
:
259
269
.
54
Xu
H.
,
Hertzel
A. V.
,
Steen
K. A.
,
Wang
Q.
,
Suttles
J.
,
Bernlohr
D. A.
.
2015
.
Uncoupling lipid metabolism from inflammation through fatty acid binding protein-dependent expression of UCP2.
Mol. Cell. Biol.
35
:
1055
1065
.
55
Furuhashi
M.
,
Tuncman
G.
,
Görgün
C. Z.
,
Makowski
L.
,
Atsumi
G.
,
Vaillancourt
E.
,
Kono
K.
,
Babaev
V. R.
,
Fazio
S.
,
Linton
M. F.
, et al
.
2007
.
Treatment of diabetes and atherosclerosis by inhibiting fatty-acid-binding protein aP2.
Nature
447
:
959
965
.
56
Xu
A.
,
Tso
A. W.
,
Cheung
B. M.
,
Wang
Y.
,
Wat
N. M.
,
Fong
C. H.
,
Yeung
D. C.
,
Janus
E. D.
,
Sham
P. C.
,
Lam
K. S.
.
2007
.
Circulating adipocyte-fatty acid binding protein levels predict the development of the metabolic syndrome: a 5-year prospective study.
Circulation
115
:
1537
1543
.

The authors have no financial conflicts of interest.

Supplementary data