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. Author manuscript; available in PMC: 2018 Jan 15.
Published in final edited form as: J Immunol. 2016 Dec 5;198(2):798–807. doi: 10.4049/jimmunol.1601403

Adipose Fatty Acid Binding Protein Promotes Saturated Fatty Acid-induced Macrophage Cell Death through Enhancing Ceramide Production

Yuwen Zhang 1, Enyu Rao 1, Jun Zeng 1, Jiaqing Hao 1, Yanwen Sun 1, Shujun Liu 2, Edward R Sauter 3, David A Bernlohr 4, Margot P Cleary 2, Jill Suttles 1, Bing Li 1
PMCID: PMC5225136  NIHMSID: NIHMS829763  PMID: 27920274

Abstract

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. Here, we report a new mechanism by which dietary FAs, in particular saturated FAs, are able to directly trigger macrophage cell death. We demonstrated that excess saturated FAs, but not unsaturated FAs, induced the production of cytotoxic ceramides in macrophage cell lines. Most importantly, expression of adipose fatty acid binding protein (A-FABP) in macrophages facilitated metabolism of excess saturated FAs for ceramide synthesis. Inhibition or deficiency of A-FABP in macrophage cell lines decreased saturated FA-induced ceramide production, thereby resulting in reduced cell death. Furthermore, we validated the role of A-FABP in promoting saturated FA-induced macrophage cell death with primary bone-marrow derived macrophages and high-fat diet-induced obese mice. Altogether, our data reveal that excess dietary saturated FAs may serve as direct triggers in induction of ceramide 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.

INTRODUCTION

Due to over nutrition and less energy expenditure, obesity in humans has reached alarming proportions (1). According to the Center for Disease Control and Prevention (CDC), about 34.9% of adults and 17% of children and adolescents are obese in the U.S. It is now clear that obesity represents a major risk factor for many co-morbid conditions, such as cardiovascular diseases, type 2 diabetes, certain types of cancers, etc (2,3). While 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). While reports demonstrate that obesity-imprinted macrophages contribute to metabolic inflammation through the production of various pro-inflammatory 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 fatty acids (also called non-esterified fatty acids, NEFA) are usually bound to plasma albumin, which usually range from 200–400 μM in normal people, but from 400–800 μM in obesity and diabetic patients (12,13). While it seems clear that saturated FAs (e.g. palmitic acid, PA and stearic acid, SA) are more lipotoxic than unsaturated FAs (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 (14–16). As lipid chaperones inside cells, fatty acid 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). While E-FABP is expressed in various immune cell types, including T cells, NK cells, B cells and macrophages (19–21), A-FABP expression is restricted to antigen presenting cells, 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 the present study, to mimic circulating excess FAs in obese conditions as reported (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 vs. unsaturated FAs. While unsaturated FAs were metabolized and stored as lipid droplets in macrophages, saturated FAs were prone to produce ceramides, leading to macrophage cell death. Most importantly, we identify A-FABP as a new molecular sensor mediating saturated FA-induced ceramide production and cell death in both immortalized and primary macrophages.

MATERIALS AND METHODS

Animals

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 (IACUC) 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 months before they were sacrificed for analysis of macrophage phenotype in the peripheral blood.

Reagents

Monoclonal anti-ceramide antibody (mouse IgM, clone MID 15B4), ROS inhibitors APDC and BHA, and triacsin C were purchased from Sigma. All caspase inhibitors were from R&D Systems and Biovision. Ceramide synthesis inhibitor fumonisin B1 and myriocin (a serine palmitoyltransferase) were from Cayman Chemical. BODIPY-C16 FA was purchased from Thermo Fisher Scientific. All flow cytometric antibodies were purchased from Biolegend. All FA sodium salts were from Nu-Chek Prep, Inc. Palmitic acid (PA, 5mM), stearic acid (SA, 5mM) oleic acid (OA, 5mM), linoleic acid (LA, 5mM) and ω-3 eicosapentaenoic acid (EPA) were prepared with 2mM endotoxin-free BSA in PBS, sonicated until dissolved and filtered through 0.2μM sterile filter.

Cell culture and treatment

Primary macrophages were differentiated from either M-CSF-induced bone marrow derived macrophages (M-BMMs) or GM-CSF-induced bone marrow derived macrophages (GM-BMMs) (24). Immortalized macrophage cell lines were established from A-FABP-deficient (A-FABP−/− macrophages) or WT mice (WT macrophages) (22,27). Briefly, bone marrow cells isolated from WT and A-FABP−/− mice were centrifuged through Fico/Lite-LM to remove red blood cells. 2×106/ml bone marrow cells were cultured in conditioned RPMI-1640 medium containing J2-CRE virus, 5μg/ml polybrene, 10ng/ml M-CSF, 10μg/ml gentamicin, 10% FBS for overnight. Non-adherent cells were collected and incubated in fresh conditioned medium as shown above for 7 days without disturbance. Virus-transformed colonies were isolated for characterization and further analysis. Macrophage cell lines (2×105 cells/ml/well) or primary BMMs were lifted and re-plated (4×105 cells/ml/well) in 24-well plates for overnight, then further treated with designated concentrations of saturated or unsaturated FAs (100, 200, and 400μM, respectively) for 18hr for further analysis. See details of M-BMM generation in the supplementary material.

Flow cytometric 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 antibodies (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 ceramide staining, macrophages stimulated with saturated or unsaturated FAs (400μM) were fixed and permeabilized with permeabilization buffer (eBiosciences) and stained intracellularly with mAb anti-ceramide antibody. All Samples were acquired on a FACS Calibur flow cytometer and data analysis was conducted using FlowJo software (Tree Star).

Confocal analysis

Macrophage cell lines were cultured on poly-D-lysine coated coverslips (NeuVitro) in a 24-well plate were treated with saturated FAs (stearic acid or palmitic acid) or unsaturated FAs (oleic/linoleic acid) for designated period of time. After fixation and permeabilization, the cells were stained with ceramide or BODIPY® 493/503(Invitrogen) as previously described (24). Nuclei were stained with 0.2μM DAPI (Invitrogen). Confocal slides were analyzed with Nikon A1 laser scanning confocal microscope.

Measurement of cell death or viability

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

Western blotting

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 BCA assay (Thermo Scientific). Anti-mouse E-FABP and A-FABP antibodies (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.

Quantitative RT-PCR

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, FATP1, FATP4, TNFα, CerS2, CerS5, CerS6. Relative mRNA levels were determined using β-actin or HPRT1 as a reference gene.

Analysis of macrophage morphology by electron microscopy

Control or saturated FA-treated macrophage cells were pelleted and fixed in 0.1M 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 pictures were examined with a transmission electron microscopy at the Research and Innovation Research Core in the University of Louisville.

SiRNA transfection

Coding region targeting DsiRNA Duplex SiRNA for A-FABP and Ceramide synthase 5 (CerS5) were ordered from Integrated DNA Technologies, Inc. To knock down gene expression, wild type BMM and macrophages (cell line) were transfected with designated SiRNA (20 to 200nM) using Oligofectamine (Life Technologies) for 24 to 48 hr before treatments.

Cytokine and ceramide species analysis

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

Statistical analysis

Student’s t test was performed for the comparison of results from different treatments. P<0.05 is considered statistically significant.

RESULTS

Saturated FAs, but not unsaturated FAs, induce macrophage cell death

Since 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. As 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 saturated FAs or unsaturated FAs. Macrophage cell death was monitored by 7-AAD staining using flow cytometry. Interestingly, saturated FAs (containing PA and SA) induced macrophage cell death in a dose-dependent manner while unsaturated FAs (containing OA and LA) exhibited no effects on macrophage survival (Figure 1A, 1B). Using a luminescent cell viability assay, we consistently observed that saturated FAs, but not unsaturated FAs, significantly decreased macrophage viability (Figure 1C). To dissect the effect of individual dietary FAs on macrophage cell death, macrophages were cultured with individual FA. As shown in Figure 1D, both PA and SA potently induced macrophage cell death while OA and LA had no or limited effects on macrophages. When we stained sFA-induced cell death using annexin V, we found majority of dying cells were annexin V-negative (Figure 1E). Transmission electron microscopy showed that dying cells lost their membrane integrity without vesicle formation (Figure 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 (EPA) appeared to induce macrophage cell death only at very high concentration (400μM) (Supplementary Figure 1). Considering the relatively low levels of ω-3 FAs in the circulation (30), saturated FAs, but not unsaturated FAs, are likely to be the major dietary FAs which induce necrotic macrophage cell death in obesity.

Figure 1.

Figure 1

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Saturated fatty acids, but not unsaturated fatty acids, induce macrophage cell death.

(A,B) Immortalized macrophages were cultured in the presence or absence of indicated total concentrations of saturated FAs (PA+SA) or unsaturated FAs (OA+LA) for 24h. Flow cytometric analysis of macrophage cell death by 7-AAD staining. The mean percentage and SD of macrophage cell death are shown in panel B. (C) Analysis of immortalized macrophage cell viability in response to indicated concentrations of saturated FAs or unsaturated FAs 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 saturated FA (400μM)-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 pictures of saturated FA-induced macrophage cell death were shown in panel F. (**p<0.01, *** p<0.001 compared to the BSA control group).

Ceramides are the major mediators of saturated FA-induced macrophage cell death

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

Figure 2.

Figure 2

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Ceramides are the major mediators of saturated fatty acid-induced macrophage cell death. (A–C) Average percentage of cell death of immortalized macrophages in response to BSA control or 400μM PA treatment with or without indicated concentrations of ROS inhibitor APDC (A), BHA (B), or triacsin C (2μM) (C) for 24h. (D,E) Histogram of intracellular staining of ceramides in immortalized macrophages in response to 400μM PA treatment with or without ceramide synthase inhibitor FB1 (10μM). Mean fluorescent intensity (MFI) of ceramides are shown in the panel E. (F) Average percentage of cell death of immortalized macrophages in response to BSA control or 400μM PA treatment with or without indicated concentrations of FB1. Data represent of three independent experiments (*p<0.05, ** p<0.01).

Long-chain FAs, either from exogenous or endogenous sources, have to be catalyzed by acyl-CoA synthetase (ACS) to form acyl-CoAs before they can be utilized in multiple metabolic pathways (34), so we used triacsin C to inhibit acyl-CoA synthetase activity to determine if PA-induced cell death can be suppressed. Triacsin C lowered the percentage of PA-induced macrophage cell death from 45% to 20% (Figure 2C), suggesting that saturated FAs are activated to produce metabolites which cause cell death. As PA could be metabolized for synthesis of ceramides (35), we showed that PA treatment induced ceramide production in macrophages, and PA-induced ceramides were able to be inhibited by fumonisin B1 (FB1), a potent inhibitor of ceramide synthases (Figure 2D, 2E). We further measured PA-induced macrophage cell death in the absence or presence of FB1 (Figure 2F). Interestingly, FB1 exhibited the similar effects as triacsin C to inhibit PA-induced cell death. Altogether, these data suggest that ceramides are the major metabolites mediating saturated FAs-induced macrophage cell death.

Saturated FAs, but not unsaturated FAs, promote ceramide production in macrophages

As unsaturated FAs were not toxic to macrophages, we speculated that unsaturated FAs did not induce ceramide synthesis in macrophages. Flow cytometric intracellular staining clearly showed that PA, but not OA or LA, significantly induced ceramide formation in macrophages (Figure 3A, 3B). To investigate why unsaturated FAs failed to induce ceramides in macrophages, we found that OA, but not PA, was largely stored in the form of lipid droplets in macrophages (Figure 3C), implying that they are sequestered and unavailable to mediate ceramide synthesis. To dissect which species of ceramides were critical for PA-induced macrophage cell death, we quantified different ceramides of macrophages in the presence or absence of PA treatment. Interestingly, PA increased the generation of all the major species of ceramides, including C16-, C18-, C20-, C22-, C24-ceramides(Cer), in macrophages (Figure 3D). Considering that C16-ceramide was the most abundant species and ceramide synthase 5 (CerS5) selectively synthesizes C16-ceramides (36), we measured the expression of ceramide synthases in macrophages and found the CerS5, but CerS2 and CerS6, was significantly upregulated in response to PA treatment (Figure 3E). We further transfected macrophages with CerS5 siRNA to knockdown CerS5 expression (Figure 3F), and found that PA-induced ceramide production (Figure 3G) and cell death (Figure 3H) was significantly inhibited when CerS5 was silenced. Taken together, our data indicate that excess saturated FAs promote ceramide synthesis in macrophages, thereby leading to cell death.

Figure 3.

Figure 3

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

Saturated FAs upregulates A-FABP expression in macrophages

To further investigate the mechanisms by which excess saturated FAs promote ceramide synthesis, we focused on the family of FABPs as they are central in regulating FA transport and coordinating lipid responses inside cells (37,38). As macrophages are notably heterogeneous and our previous studies demonstrated that 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 Figure 4A and 4B, the macrophage cell line exhibited a CD36+ CD11b+ Ly6c−F4/80highCD11c− phenotype with high expression of both A-FABP and E-FABP, which were different to GM-CSF-induced bone marrow derived macrophages (GM-BMMs) with a CD36−CD11b+ Ly6c−F4/80lowCD11c+ 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 (Figure 4C). Western blotting further confirmed the upregulation of A-FABP expression by PA at the protein level (Figure 4D), implying a critical role of A-FABP in mediating saturated FA-induced lipid responses in this phenotype of macrophages.

Figure 4.

Figure 4

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Saturated fatty acids 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 antibody staining). (B) Real time PCR analysis of expression of FABP family members in immortalized macrophages. (C, 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 15h. Data are representative of three independent experiments.

A-FABP expression promotes ceramide production in macrophages

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

Figure 5.

Figure 5

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PA-induced ceramide 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, C) Intracellular staining of PA-induced ceramide production in WT macrophage cell line transfected with scramble or A-FABP specific siRNA. MFI of ceramide was shown in panel 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 ceramide production in WT and A-FABP−/− macrophage cell lines after treatment with PA, SA, or OA treatment (400μM) for 18h by intracellular flow cytometric staining. (F-M) Measurement of levels of C14-ceramide(Cer) (F), C16-Ceramide (G), C18-ceramide (H), C-20 ceramide (I), C22-ceramide (J), S1P (K), Sphinganine (L), and C24-1 ceramide (M) by in WT and A-FABP−/− macrophage cell lines treated with PA for 18h by mass spectrometry. Data are representative of three independent experiments (*, p<0.05; **, p<0.01).

A-FABP deficiency protects macrophage against saturated FA-induced ceramide-mediated cell death

To further investigate the role of A-FABP in saturated FA-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 above ceramide data, treatment with OA did not induce macrophage cell death regardless of A-FABP expression (Figure 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 Figure 3A). Moreover, dual staining for ceramide+ and 7-AAD+ dead cells with flow cytometry clearly showed that A-FABP deficiency specifically inhibited saturated FA-induced ceramide+ dead cells (Q2 population), but not ceramide− dead cells (Q1 population) (Figure 6B), indicating a specific role of A-FABP in promoting ceramide-mediated cell death. To determine how A-FABP promoted saturated FA-induced cell death, we found that A-FABP deficiency neither impacted the expression of major FA transport proteins (FATPs) (Supplementary Figure 3B, 3C), nor affected FA uptake by macrophages (Figure 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 (Figure 6D, 6E). These data suggest that A-FABP deficiency may not impact lipid uptake, but rather restricting lipid utilization, thereby lowering ceramide synthesis in macrophages.

Figure 6.

Figure 6

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

A-FABP deficiency inhibits saturated FA-induced cell death in primary macrophages and obese mouse models

After demonstrating an essential role of A-FABP in mediating saturated FA-induced cell death in macrophage cell lines, we further validated these findings using primary bone marrow-derived macrophages. As compared to GM-CSF induced bone marrow derived macrophages (GM-BMMs), M-CSF induced bone marrow derived macrophages (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 (Figure 7A). More importantly, M-BMMs had higher levels of A-FABP than GM-BMMs at both RNA levels and protein levels (Figure 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 to macrophage cell lines, A-FABP deficiency in BMMs reduced cell death induced by PA by approximately 50% (Figure 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 (Figure 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 Figure 4A), nor the percentage and phenotype of monocytes in the bone marrow (supplementary Figure 4B, 4C). However, the percentage of peripheral CD36+ monocytes in obese A-FABP−/− mice was significantly higher than these in obese WT mice (supplementary Figure 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 fatty acid-induced cell death, thus contributing to the elevated A-FABP+ CD36+ monocyte subsets in obese A-FABP−/− mice.

Figure 7.

Figure 7

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

DISCUSSION

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 remains 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). Herein, we report when various dietary FAs were up-taken by stable macrophage cell lines or primary bone-marrow derived macrophages, only saturated FAs (e.g. PA, SA) were metabolized to produce ceramides to induce macrophage cell death. Most importantly, we identified A-FABP as a new molecular sensor in mediating excess saturated FAs-induced ceramide production and in promoting macrophage cell death, thus contributing to the sterile chronic inflammation in obesity.

In the present 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), whether upregulated levels of fatty acids 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 saturated FA (e.g. PA and SA), but not unsaturated FAs (e.g. OA and LA), induced significant cell death after overnight culture. These results are consistent with other studies using myocytes or hepatocytes (42,43). In determining the specific effects of saturated FAs in inducing macrophage cell death, we found that saturated FAs are metabolized to produce cytotoxic ceramides in macrophages, which can be inhibited either by silencing CerS5 or by FB1, a specific ceramide synthase inhibitor. In contrast, unsaturated FAs are sequestered in lipid droplets averting away ceramide pathways. Thus, our data reveal saturated FAs as new sterile stimuli inducing macrophage cell death. Notably, cytotoxic effects of saturated FAs have been shown in various types of cells, including heart-tissue derived H9C2 cells and ovary cells, through generation of ROS (44,45). However, ROS did not appear to be a major mechanism in mediating saturated FA-induced cell death in macrophages in that 1) saturated FA-induced macrophage cell death started early in 12h before iNOS was majorly produced, 2) both non-toxic OA and toxic PA induced equivalent amounts of iNOS production in macrophages, 3) ROS specific inhibitors, including APDC and BHA, exhibited only marginal effects on PA-induced macrophage death, which was consistent to previous studies showing ceramide-induced, ROS-independent necrosis in Jurkat and U937 cells (46,47). Instead, in response to environmental stimuli, macrophages produced large amounts of ROS for maintaining immune surveillance and tissue homeostasis (48,49). Moreover, ROS produced by macrophages can even promote their survival and differentiation (48,49). Thus, compared to non-phagocytic cells, phagocytic macrophages exhibit unique mechanisms in response to excess saturated FAs in the setting of obesity.

Secondly, 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 induced by M-CSF exhibit CD11b+F4/80+Ly6C−CD11c−CD36+ phenotype (M2-like) while GM-BMMs induced by GM-CSF are CD11b+F4/80+Ly6C−CD11c+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 saturated FAs (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 (50). 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.

Thirdly, 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 to other FABPs was specifically upregulated in M-GMMs, suggesting a unique role of A-FABP in meditating lipid metabolism in these cells (51). In line with our observations, A-FABP has been shown to be positively associated with CD36 expression by activated PPARγ in macrophages (52–54). Our previous studies have shown that E-FABP, but not A-FABP, is highly expressed CD11c+ CD36− macrophages in different settings of diseases (24,55). 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 type 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), thus may reduce FA-mediated cytotoxic metabolic pathways. This may explain why CD36− macrophages are more tolerant than CD36+ macrophages in response to saturated FA-induced cell death.

Lastly, to determine whether A-FABP is essential in mediating saturated FA-induced ceramide 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 saturated FA-induced ceramide production and cytotoxicity. Further analyses indicate that A-FABP deficiency affects neither FA uptake, nor expression of ceramide synthases 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 (56). Using bone marrow-derived primary macrophages (M-BMMs), although exhibiting relative resistance to PA-treatment as compared to SA-treatment, we demonstrated that A-FABP deficiency significantly inhibited both saturated FA-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 saturated FA-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 very lowly expressed in immature bone marrow cells. We also noticed that neither inhibition of ceramide synthesis, nor deficiency of A-FABP expression, was able to completely inhibit saturated FA-induced macrophage cell death, suggesting that other signaling pathways are also involved in saturated FA-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 (57,58), and provide mechanistic evidence by establishing A-FABP as a new molecular sensor for saturated FA-induced ceramide synthesis and cell death of macrophages.

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

Supplementary Material

1

Acknowledgments

We thank Dr. Xuan-Mai T. Person and Dr. Michael D. Jensen for ceramide species analysis with lipid mass spectrometry. We thank Dr. Brian Watterberg for his 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 NIH R01 grants (CA18098601A1, CA17767901A1), NIDDK (U24DK100469), and NCATs (UL1TR000135).

Footnotes

Disclosure of Potential Conflicts of Interest

The authors state no conflict of interest.

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