Induction of fatty acid synthesis is a key requirement for phagocytic differentiation of human monocytes

Abstract

Monocytes are precursors of macrophages. Here we demonstrate that macrophage colony-stimulating factor (M-CSF)-dependent differentiation of primary human monocytes from healthy volunteers induces transcription of SREBP-1c target genes required for fatty acid (FA) biosynthesis and impairs transcription of SREBP-2 target genes required for cholesterol synthesis. Detailed lipid metabolic profiling showed that this transcriptional regulation leads to a dramatically increased fatty acid synthesis as driving force for enhanced phospholipid synthesis. During cell differentiation the major lipid class switches from cholesterol in monocytes to phosphatidylcholine in macrophages. Ultrastructural analysis revealed that this transcriptional and metabolic regulation is essential for development of macrophage filopodia and cellular organelles including primary lysosomes, endoplasmic reticulum, and Golgi network. Additional functional studies showed that suppression of fatty acid synthesis prevents phagocytosis representing a central macrophage function. Therefore induction of fatty acid synthesis is a key requirement for phagocyte development and function.

Macrophages are multifunctional cells of the innate immune system present in all tissues in the body. They participate in numerous biological processes ranging from tissue and organ development to bone remodelling and wound healing (1). A hallmark of macrophage function is their phagocytic capacity (2). Differentiation of monocytes into macrophages is initiated by macrophage colony-stimulating factor (M-CSF). M-CSF (also known as CSF-1) circulates at nanomolar levels in plasma and is constitutively generated by several cell types. M-CSF levels are elevated at sites of inflammation in pathological states including autoimmune disorders, vasculitis, arthritis, and obesity (3).

Investigation of mechanisms underlying monocyte differentiation is central to the understanding of fundamental macrophage biology and metabolic diseases. Although a link between peroxisome proliferator-activated receptor-γ (PPARγ)-dependent inflammation and monocyte differentiation has been found (4, 5), little is known about the role of lipid synthesis and almost none about the role of fatty acid (FA) metabolism for monocyte differentiation.

Major regulators of lipid homeostasis in mammalian cells are the nuclear transcription factors sterol regulatory element-binding proteins (SREBPs), which belong to the family of basic helix loop helix leucine zipper (bHLH-LZ) transcription factors. SREBP-2 mainly controls genes of the cholesterol pathway and SREBP-1c preferentially regulates genes involved in FA biosynthesis (6). A well-characterized SREBP-1c target gene and key enzyme required for endogenous FA synthesis in mammalian cells is fatty acid synthase (FAS), because it catalyzes the generation of palmitate from acetyl-CoA (7). Palmitate is either elongated to stearate by long chain fatty acid elongase 6 (ELOVL6) (8) or desaturated to palmitoleate by stearoyl-CoA desaturase (SCD) (9). Fatty acid desaturase 1 (FADS1) and FADS 2 are SREBP-1c-regulated desaturases for polyunsaturated fatty acids (PUFAs) (10).

In this study we identified a massive up-regulation of genes required for FA synthesis during M-CSF–dependent differentiation of primary monocytes from healthy volunteers. We further demonstrate an essential role of FA and phospholipid synthesis for the morphogenic alterations during phagocytic differentiation of monocytes, including organelle development and macrophage function.

Results

Transcriptional Activation of SREBP-1c Target Genes and Inverse Regulation of SREBP-2 Target Genes During Monocyte Differentiation.

Global gene expression analysis of primary human M-CSF differentiated monocytes from healthy donors with DNA microarrays revealed a significant induction of SREBP-1c and a down-regulation of SREBP-2 target gene expression (Table S1). To validate these results, gene expression was analyzed with qRT-PCR of undifferentiated monocytes and for 1, 4, or 6 days with M-CSF differentiated monocytes. We could verify the induction of SREBP-1c regulated genes during monocyte differentiation (Table 1). FAS, ELOVL6, and SCD were massively up-regulated when M-CSF-dependent cell differentiation was induced. In sharp contrast, expression of genes involved in cholesterol metabolism remained unchanged or was only moderately increased when monocyte differentiation was induced. During later differentiation stages transcription of SREBP-2 target genes was down-regulated (except for PMVK, LSS, and DHCR7).

Table 1.

Up-regulation of SREBP-1c target genes and down-regulation of SREBP-2 target genes during monocyte differentiation

Gene/pathway	Symbol	Mean change	Mean change	Mean change
SREBP-1c target genes: fatty acid synthesis		Mac(d1)	Mac(d4)	Mac(d6)
Sterol regulatory element binding protein 1	SREBP-1c	1.36	8.38	6.43
Acetyl-CoA carboxylase alpha	ACACA	0.97	1.26	2.01
Fatty acid synthase	FAS	3.05	11.32	4.42
Elongase of long chain fatty acids 6	ELOVL6	12.27	131.04	74.72
Stearoyl-CoA desaturase 1	SCD1	3.95	58.42	39.26
Fatty acid desaturase 1	FADS1	1.42	6.29	2.75
Fatty acid desaturase 2	FADS2	2.48	12.93	6.72
SREBP-2 target genes: cholesterol synthesis
Sterol regulatory element binding protein 2	SREBP-2	1.33	1.25	0.67
Acetyl-CoA acetyltransferase 2	ACAT2	1.56	1.87	0.87
3-Hydroxy-3-methylglutaryl-CoA reductase	HMGCR	1.96	1.10	0.41
3-Hydroxy-3-methylglutaryl-CoA synthase 1	HMGCS1	1.77	0.73	0.31
Mevalonate kinase	MVK	1.13	1.12	0.35
Phosphomevalonate kinase	PMVK	1.26	1.68	1.44
Mevalonate decarboxylase	MVD	0.96	0.69	0.38
Geranylgeranyl diphosphate synthase 1	GGPS1	0.7	0.62	0.62
Isopentenyl-diphosphate delta isomerase 1	IDI1	1.72	1.08	0.52
Farnesyl-diphosphate farnesyltransferase 1	FDFT1	1.22	0.85	0.29
Squalene epoxidase	SQLE	1.95	1.29	0.55
Lanosterol synthase	LSS	1.99	5.03	3.26
Cytochrome P450 (51A1)	CYP51A1	1.67	1.20	0.57
Sterol-C5-desaturase	SC5DL	1.73	1.33	0.83
7-dehydrocholesterol reductase	DHCR7	4.19	11.52	2.72
Low density lipoprotein receptor	LDLR	1.57	0.83	0.35

Expression of SREBP-1c and SREBP-2 target genes in macrophages differentiated with M-CSF for 1, 4, or 6 days compared with monocytes, analyzed with qRT-PCR; 18S rRNA was used as reference gene. The boldface type indicates that the change is ≥2 or ≤0.5.

Induced Fatty Acid Synthesis and Desaturation During Monocyte Differentiation.

To test whether up-regulation of SREBP-1c target genes has an impact on macrophage lipid composition, FA profiles were analyzed during monocyte differentiation (Fig. 1A). At day 4 of M-CSF-mediated differentiation, we could detect a striking shift of FA composition from saturated and polyunsaturated to monounsaturated FAs. The C16 and C18 monounsaturated FA content increased from 15% in monocytes to 38% in macrophages.

Fig. 1.

Increased FA, PC, and PE synthesis during monocyte differentiation. (A) FA profile during monocyte differentiation, analyzed by GC-MS. (B) Induced FA synthesis in macrophages (d1, d4, and d6). (C) Increased FA desaturation in macrophages (d4 and d6). (D) Increased FA elongation in macrophages (d1, d4, and d6). (E) Free cholesterol (FC) is the predominant lipid in monocytes, whereas phosphatidylcholine (PC) is the major lipid in macrophages (d4 and d6). PC, sphingomyelin (SM), dihydrosphingomyelin (Dih-SM), phosphatidylethanolamine (PE), PE–based plasmalogenes (PE-pl), phosphatidylserine (PS), lysophosphatidylcholine (LPC), FC, and cholesteryl ester (CE) were quantified by ESI-MS/MS. (F) Decreased cholesterol synthesis in macrophages (d1, d4, and d6). (G and H) Increase of D₉-PC and D₄-PE synthesis during monocyte differentiation. (I and J) After 4 h stable isotope labeling ¹³C₂-PE and ¹³C₃-PS synthesis do not show considerable differences between monocytes and macrophages. (*, P < 0.05; **, P < 0.01; ***, P < 0.001)

In a next step, we asked whether the changes in FA profiles are due to increased activities of the enzymes FAS, SCD, and ELOVL6 (Fig. S1). To profile FA synthesis, cells were incubated with stable isotope-labeled acetate and incorporation into palmitate was monitored (Fig. S1). In monocytes we observed almost no FA synthesis, whereas induction of M-CSF-dependent differentiation stimulated palmitate synthesis 28-fold (Fig. 1B). To explore FA desaturation and elongation, cells were supplied with stable isotope-labeled palmitate and conversion to palmitoleate and stearate was determined (Fig. S1). Palmitate desaturation was not detectable in monocytes, but in macrophages (Fig. 1C). Desaturation ratios increased 25-fold from day 1 to day 6 of differentiation, whereas palmitate elongation revealed only a modest rise during monocyte differentiation (Fig. 1D). Taken together these lipid species data are in good agreement with the transcriptional results. The shift to monounsaturated C16 and C18 FAs in macrophages results from a strong induction of FA biosynthesis and desaturation during cell differentiation.

Enhanced Fatty Acid Synthesis Increases Glycerophospholipid Content During Monocyte Differentiation.

Next, we explored lipid composition of cells during differentiation. With 28% of the analyzed lipids, cholesterol is the dominating lipid of monocytes followed by phosphatidylcholine (PC) with 24% (Fig. 1E). Consistent with impaired SREBP-2 target gene expression in macrophages, the free cholesterol (FC) fraction declined during cell differentiation to 10%. Moreover, analysis of stable isotope-labeled acetate incorporation in FC during cell differentiation showed a decreased FC synthesis (Fig. 1F). The PC content dramatically increased to 40% becoming the major lipid class of macrophages. Phosphatidylethanolamine (PE + PE-plasmalogens) content was elevated, whereas phosphatidylserine (PS) levels were lowered. Overall the content of PC and ethanolamine-containing phospholipids rose from 40 to almost 70%. Analysis of the species pattern of the individual glycerophospholipid (PL) classes revealed a remarkable shift from the longer and more unsaturated to shorter and less unsaturated PL species during macrophage differentiation (Fig. S2).

To investigate glycerophospholipid biosynthesis, we supplied cells with stable isotope-labeled choline (D₉), ethanolamine (D₄), and serine (¹³C₃) for 4 h and 24 h (Fig. S1). Monocytes and macrophages at day 1 showed marginal de novo PC and PE synthesis (Fig. 1 G and H). By contrast, at day 4 of differentiation D₉-PC synthesis was found increased almost 10-fold and D₄-PE synthesis 3-fold. The species pattern of newly synthesized glycerophospholipids changed in a similar way to the total species pattern (Fig. S2). In summary, the increase of PC and PE content during macrophage differentiation corresponds very well to the increased glycerophospholipid synthesis rates. The shift to shorter and less unsaturated glycerophospholipid species during phagocytic differentiation of monocytes is in good agreement with up-regulation of FA synthesis and desaturation resulting in an increased fraction of C16 and C18 saturated and monounsaturated FAs.

Phosphatidylcholine and Phosphatidylethanolamine Synthesis Are Fatty Acid Synthesis-Dependent in Macrophages.

To show a direct connection of FA and PL synthesis during differentiation of monocytes, we first inhibited enzymes required for FA synthesis by blocking SREBP processing with 25-hydroxycholesterol (25-HC) (11). 25-HC dose dependently reduced transcription, protein levels, and enzyme activities of the SREBP-1c target genes FAS and SCD (Fig. 2A and Fig. S3). We found that decreased FA synthesis (Fig. 2B) led to decreased PC (about 65% of control) (Fig. 2C) and PE synthesis (about 75% of control) (Fig. 2D).

Fig. 2.

Up-regulation of FA synthesis is coupled to PC and PE synthesis (A) Treatment with 25-HC reduces mRNA expression of SREBP-1c target genes FAS and SCD, and increases LXRα transcription in macrophages (d4). (B) FA synthesis is inhibited by 25-HC and induced LXR stimulation (2.5 μM T0901317, 24 h) in macrophages (d4). (C and D) FA synthesis (25-HC)-dependent modulation of PC and PE synthesis. (E) FA synthesis is inhibited by cerulenin in macrophages (d4). (F and G) Inhibition of FA synthesis decreased PC and PE synthesis and is rescued by addition of FA-CoA mix (5 μM FA 16:0-CoA, 2 μM FA 16:1-CoA, 4 μM FA 18:0-CoA, 4 μM FA 18:1-CoA, and 2 μM FA 18:2-CoA for 24 h). (H and I) Decreased SREBP-1c and FAS expression in primary cells treated for 48 h with siRNAs. (J) FA synthesis is inhibited by siRNAs against SREBP-1 and FAS. (K and L) SREBP-1 and FAS-dependent modulation of PC and PE synthesis. (*, P < 0.05; **, P < 0.01; ***, P < 0.001)

As a positive control we enhanced FA synthesis by SREBP-1c activation through the LXR-stimulation agent T0901317 (12). LXR activation increased FA synthesis (Fig. 2B), D₉-PC (Fig. 2C) and D₄-PE (Fig. 2D) generation (140% and 120%). However, it was not possible to increase FA and D₉-PC synthesis of 25-HC-treated macrophages by LXR stimulation, implying that LXR is not able to induce FAS levels independently from SREBP. Moreover, elevated LXRα levels observed under 25-HC treatment (Fig. 2A and Fig. S3) could not restore FA synthesis, arguing for an indirect action of LXR on FAS levels via SREBP induction.

As 25-HC inhibits both SREBP-1c and SREBP-2 processing and thus FA and cholesterol synthesis (11), we next suppressed FA synthesis with cerulenin and C75 (Fig. 2E and Fig. S4), well-characterized and specific inhibitors of FAS (13, 14). Similar to 25-HC, cerulenin and C75, reduced D₉-PC and D₄-PE synthesis (Fig. 2 F and G and Fig. S4). Most importantly, the cerulenin- and C75-dependent decrease of PL synthesis was rescued by addition of a FA-CoA mix composed of the FAs generated during cell differentiation.

To further strengthen our findings we next used a gene-specific targeting approach to knock down fatty acid synthesis. Primary monocytes were transfected with a nontargeting control siRNA or siRNAs against SREBP-1 or FAS. qRT-PCR analysis showed a drop of SREBP-1c and FAS mRNA expression by about 55% and 70%, respectively (Fig. 2 H and I and Fig. S6). Both SREBP-1 and FAS knockdown reduced fatty acid synthesis to 40% (Fig. 2J) and were accompanied by suppressed D₉-PC and D₄-PE synthesis (Fig. 2 K and L).

In summary, these data show that during phagocytic differentiation of monocytes the up-regulation of PC and PE synthesis is clearly linked to FA synthesis.

Modulation of Fatty Acid Synthesis Alters Macrophage Ultrastructure and Organelle Development.

Next we asked whether inhibition of FA synthesis influences macrophage ultrastructure. It is known that monocyte differentiation increases the volume of the cells and the number of intracellular organelles (15 –17). Accordingly, electron microscopy showed monocytes as small round cells with short filapodia and a big nucleus (Fig. 3A). The cytoplasm contained few primary lysosomes, mitochondria, and short endoplasmatic reticulum (ER) sections. In contrast, macrophages (day 4) had about three times the size of monocytes with numerous and long filopodia (Fig. 3B). Their cytoplasm was packed with organelles including mitochondria, primary lysosomes, long ER sections, and particularly large Golgi complexes (Fig. 3C).

Fig. 3.

Modulation of FA synthesis alters macrophage ultrastructure and organelle development. Ultrastructural analysis was performed with transmission electron microscopy. (Scale bar, 0.5 μm.) Endoplasmic reticulum (ER), lysosome (Ly), mitochondrium (M), nucleus (N). A, B, D, and E are scaled equally. Relative sizes and organelle content were estimated from multiple EM pictures for each condition. (A) Monocytes are small round cells with short filopodia and a big centered nucleus. (B) Macrophages (d4) have about three times the size of monocytes, containing numerous and long filopodia and their cytoplasm is packed with organelles (Golgi, ER, Ly, and M massively increase). (C) Magnification from B highlighting the large Golgi complexes of macrophages. (D) Inhibition of lipid synthesis [2.5 μM 25-HC, Mac(d4)] decreases cell size (by ~40%), filopodia, and cellular organelles (by ~70% for M, by ~80% for Ly; Golgi is missing, only short ER sections). (E) Inhibition of FAS [2 μg/mL cerulenin, Mac(d4)] decreases cell size (by ~40%), filopodia, and cellular organelles (by ~50% for M, by ~70% for Ly, by ~50% for Golgi, short ER sections).

When 25-HC was present the cells were smaller with less and shorter filopodia than untreated macrophages, even though they were not as small as monocytes (Fig. 3D). The cells had only a few mitochondria and short ER sections; the Golgi complex, when existent at all, was small. Overall upon addition of 25-HC, the cells appeared rather like monocytes than macrophages. Similarly, suppression of FAS with cerulenin led to smaller cells with fewer and less developed filopodia and organelles (Fig. 3E). After addition of FA-CoAs, 25-HC-treated cells increased their Golgi complex and number of lysosomes; addition of FA-CoAs to cerulenin-treated macrophages led to an increase of filopodia, lysosomes, and mitochondria (Fig. S5). Macrophages, where SREBP-1 and FAS was knocked down with RNAi, had only half of the size of cells treated with a control siRNA (Fig. S6).

Taken together, these data show that modulation of FA synthesis during cell differentiation massively affects macrophage ultrastructure. Suppression of FA synthesis prevents development of filopodia and cellular organelles including mitochondria, primary lysosomes, ER, and the Golgi complex.

Induction of Fatty Acid Synthesis Is Crucial for Monocyte Differentiation and Phagocytic Activity of Macrophages.

Finally, we asked whether inhibition of lipid synthesis affects monocyte differentiation to macrophages and their key function as phagocytes (2). Chitotriosidase (CHIT1) and human cartilage 39-kDa glycoprotein (CHI3L1) are late macrophage differentiation markers (18, 19). A SREBP-dependent regulation of CHIT1 and CHI3L1 has not yet been described. CHIT1 expression is controlled by the transcription factors C/EBPβ and PU.1; CHI3L1 is regulated by NFκB (20, 21).

As expected, mRNA expression analysis revealed a dramatic increase of CHI3L1 and CHIT1 expression during macrophage development (Table S2). Addition of 25-HC massively lowered CHI3L1 and CHIT1 expression (Fig. 4 A and B). Similarly, inhibition of FA synthesis with cerulenin and C75 reduced expression levels of these late differentiation markers (Fig. 4 A and B and Fig. S4). Importantly, expression of CHI3L1 and CHIT1 could be restored partially in cerulenin- and C75-treated cells by mix of C16 and C18 FA-CoAs. To provide more evidence that macrophage differentiation depends on fatty acid synthesis, the macrophage differentiation markers CD11b, CD36, and MRC1 (mannose receptor) were analyzed in cells treated with siRNAs against SREBP-1 or FAS (22 –24). Knockdown of FA synthesis with RNAi was accompanied by a lowered cell-surface expression of CD11b, CD36, and MRC1 (Fig. 4C).

Fig. 4.

Induction of FA synthesis is crucial for phagocytic differentiation and function. (A and B) Suppression of CHI3L1 and CHIT1 expression by inhibition of FA synthesis and rescue by addition of FA-CoA mix, analyzed with qRT-PCR. (C) Decreased surface expression of CD11b, CD36, and MRC1 (CD206) of primary macrophages treated for 48 h with siRNAs, analyzed by flow cytometry. (D) Inhibition of FA synthesis reduced phagocytosis of macrophages (d4), which is rescued by addition of FA-CoA mix, analyzed by flow cytometry. (E) Knockdown of SREBP-1 and FAS suppresses phagocytosis of primary macrophages. (*/^#, P < 0.05; **/^##, P < 0.01; ***/^###, P < 0.001. *, P values were calculated relative to untreated samples; ^#, P values were calculated relative to cerulenin- or 25-HC-treated samples.)

Phagocytic activity of macrophages was investigated by uptake of fluorescent phagobeads. The addition of 25-HC, cerulenin, and C75 decreased macrophage phagocytosis, respectively (Fig. 4D and Fig. S4). Phagocytosis could be rescued partly (25-HC) or completely (cerulenin) when cells were supplemented with a FA-CoA mix. siRNAs against SREBP-1 and FAS decreased macrophage phagocytosis by at least 90% compared to cells transfected with a control siRNA (Fig. 4E). Finally, we explored FA-synthesis-dependent uptake of enzymatically modified LDL (E-LDL), a lipoprotein ingested by macrophages through phagocytosis and found in advanced atherosclerotic lesions (25). Again, inhibition of FA synthesis resulted in reduced cellular cholesterol levels, indicating an impaired uptake of E-LDL (Fig. S7).

In summary, these results show that induction FA synthesis is absolutely necessary for macrophage differentiation and function. Inhibition of FA synthesis prevents phagocytic capacity of the cells.

Discussion

A basic feature of monocytes and a crucial step during atherosclerosis development is their ability to differentiate into phagocytes. Although several groups investigated alteration of gene expression associated with monocyte differentiation through global transcriptional profiling approaches (26), the role of lipid synthesis in this process is poorly understood. Transcriptional data of more than two stages of cell differentiation are rare and lipid metabolic and functional data are completely unavailable.

We found an inverse transcriptional regulation of SREBP-1c and SREBP-2 target genes leading to strongly induced fatty acid synthesis and a decreased cholesterol fraction during phagocytic differentiation of monocytes. Besides decreased cholesterol synthesis, the lower cholesterol fraction during cell differentiation might also be due to LXR-mediated induction of the cholesterol transporters ABCA1 and ABCG1 (Table S2) and increased cholesterol efflux. Extensive characterization of monocyte and macrophage lipid metabolism showed that up-regulation of FA synthesis during cell differentiation is the driving force for enhanced PC and PE synthesis. Importantly, induction of FA and phospholipid synthesis is independent of serum in the macrophage cultivation media, because addition of serum during differentiation does not decrease lipid synthesis (Fig. S8).

Because we did not observe a transcriptional increase of genes directly involved in PC and PE biosynthesis (Table S2), either a posttranscriptional regulation of these enzymes or a metabolic activation (through an enlarged fatty acid pool) of PL synthesis may be concluded. Choline-phosphate cytidyltransferase (CCT) (Fig. S1, PCYT1), the rate-limiting enzyme of de novo PC synthesis, is mainly regulated on the posttranscriptional level (27 –29). Upon activation, its soluble inactive form is translocated to the membrane, which is facilitated by electrostatic interactions with negatively charged membrane lipids, for instance FAs. This conclusion is in good agreement with a study in CHO cells, which showed that increased PC synthesis is mainly due to induced fatty acid synthesis rather than a transcriptional activation of CCT (30).

The down-regulation SREBP-2 target genes during phagocytic differentiation of monocytes was accompanied by a decreased FC fraction. Together with an increase in PL content due to enhanced de novo synthesis the FC/PL ratio dramatically dropped from 0.5 in monocytes to 0.1 in macrophages. Cholesterol is one of the most important regulators of lipid organization decreasing membrane fluidity (31). Thus, a lower FC/PL ratio in macrophages results in more fluid and flexible membranes that support phagocytosis. Therefore PL synthesis is absolutely necessary for macrophage function, which is also confirmed by the finding that cytokine secretion of macrophages requires PC synthesis (32).

More than half of the PE-containing lipids in macrophages are PE-pl, which are known to protect macrophages from oxidative stress by their ability to bind and scavenge free radicals (33). Concerning cellular damage, a low FC/PL ratio in macrophages may also be an adaptive precaution of cells to protect from cholesterol-mediated cytotoxicity during uptake of modified lipoproteins. High free cholesterol levels are known to form cytotoxic cholesterol crystals (34). Hence, mature macrophages may build a “buffer” for further cholesterol uptake, with an enhanced PL content through high PL synthesis. Blocking of PC synthesis during FC loading results in enhanced death of macrophages (35).

Analyzing cell morphology during cell differentiation showed that macrophages strongly increase their size and number of intracellular organelles including the Golgi complex, ER, and primary lysosomes. We found that modulation of fatty acid synthesis massively impacts cell size, filopodia, and cellular organelle development. In mammalian cells, the FC/PL ratio of the plasma membrane is 1.0, whereas membranes of the Golgi complex and the ER have a FC/PL ratio of 0.2 and 0.15, and the FC/PL ratio of lysosomal membranes is 0.38 (36, 37). Thus these organelles have very low amounts of cholesterol in their membranes; their main membrane lipids are the phospholipids PC and PE (36), which fits very well with the up-regulation of phospholipid synthesis during cell differentiation and the change or their lipid composition. Importantly, the ER is a source of membrane for phagosome formation and therefore expansion of ER is a prerequisite for phagocytosis in macrophages (38, 39). In our experiments inhibition of FA synthesis resulted in less ER, which might also contribute to a reduced phagocytosis in these cells.

Beside biophysical and metabolic effects, an increased FA pool may also influence nuclear hormone receptor activity in macrophages. PPARs are well-known sensors of fatty acids (40). Induction of PPARγ promotes the uptake of oxidized LDL through transcriptional induction of the scavenger receptor CD36 (5) and is required macrophage activation (4, 41). This might also explain our finding that the uptake of modified lipoproteins is inhibited when fatty synthesis is suppressed during the differentiation process.

In summary, we could identify an inverse regulation of FA/phospholipid and FC synthesis during phagocytic differentiation of primary human monocytes leading to a decreased FC/PL ratio. This adaptation of lipid metabolism is required for cellular organelle development and macrophage function. Consequently, our results suggest a previously unknown role for FA synthesis in the phagocytic differentiation of monocytes, which may be important for tissue development, host defense, and in human metabolic diseases such as atherosclerosis.

Materials and Methods

Reagents and Materials.

The 2-¹³C-acetate, ¹³C₃-serine, D₃-palmitate, D₄-ethanolamine, and D₉-choline were obtained from Cambridge Isotope Laboratories. 25-HC, cerulenin, C75, T0901317, and fatty acid-CoAs were purchased from Sigma-Aldrich.

Monocyte Isolation and Cell Culture.

Primary human monocytes were obtained from healthy donors by leukapheresis and counterflow elutriation as described previously (42). Cells were cultured on plastic Petri dishes in macrophage SFM medium (Gibco-BRL) and allowed to differentiate for 1, 4, or 6 days in the presence of 50 ng/mL recombinant human MCSF from R&D Systems.

RNA Isolation and Quantitative RT-PCR Analysis.

Total RNA was extracted with the RNeasy Midi Kit (Qiagen); cDNA was generated using the Reverse Transcripton System from Promega. Real-time quantitative RT-PCR analysis was performed with an ABI7900HT machine (Applied Biosystems). Transcripts have been specified with predesigned and optimized Assays on Demand (Applied Biosystems); 18S rRNA was used as reference gene. Relative quantification was carried out with the SDS 2.3 software (Applied Biosystems).

Protein Isolation and Analyses.

Total cell lysates were prepared in RIPA buffer (Roche). Samples with equal amounts of protein were separated on SDS gels, transferred on PVDF membranes, and incubated with anti-β-actin (Sigma-Aldrich), anti-FAS (Assay Design), anti-SREBP-1 antibodies (Santa Cruz) with a dilution of 1:1,000 for 2 h. Peroxydase-conjugated goat anti-mouse IgG (Dianova) were used at 1:20,000 for 1 h and visualized with chemoluminescence on film (ECL Plus, Amersham Pharmacia Biotech).

RNAi Experiments.

Freshly isolated human monocytes were obtained from healthy volunteers; subsequently 1 million cells were electroporated with 50 nM siRNA using the Human Monocyte Nucleofector Kit (Amaxa) and seeded in 6-well plates with macrophage medium containing MCSF for 48 h. All siRNAs were “human validated siRNAs” from Ambion; the sequence of the nontargeting control siRNA was 5′-AGUACUGCUUACGAUACGGTT-3′, the sequence of the SREBP-1 siRNA was 5′-GGCAAAGCUGAAUAAAUCUTT-3′, and the sequence of the FAS siRNA was 5′-GGUAUGCGACGGGAAAGUATT-3′.

Fatty Acid Analysis.

Total FA analysis was carried out by GC-MS (for details, see Fig. S1).

Lipid Analysis.

Lipids were extracted according to the procedure described by Bligh and Dyer (43) in the presence of nonnaturally occurring lipid species as internal standards. Lipids were quantified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in positive ion mode as described previously (44) (for details, see Fig. S1).

Ultrastructural Analysis.

Ultrastructural analysis was performed with transmission electron microscopy with standard protocols as described previously (45).

Phagocytosis Assays and Other FACS Analysis.

Phagocytic capacity was determined with flow cytometry. Primary macrophages were incubated with 1.1 × 10¹¹ phagobead particles/mL (fluoresbrite yellow green microspheres, 0.75 μm, Polysciences Europe GmbH); 2 h later, fluorescence was determined with a FACS Calibur flow cytometer (BD Biosciences). Expression of CD11b, CD36, and MRC1 (CD206) was determined by flow cytometry (1 × 10⁵ cells per analysis) with antibodies from BD Biosciences.

Statistics.

The level of significance for the difference between data sets was assessed using Student's independent t test (*/^#, P < 0.05; **/^##, P < 0.01; ***/^###, P < 0.001). *, P values were calculated relative to control samples; ^#, P values were calculated relative to cerulenin- or 25-HC–treated samples. Results are expressed as mean ± SEM.