Fatty Acid Composition, at Equivalent Lipid Exposure, Dictates Human Macrophage Polarization via PPARγ Signaling

Abstract

Dietary fats are consumed as mixtures, yet it remains unclear whether fatty acid composition, independent of fat content, dictates human macrophage polarization. We compared two defined mixtures containing identical fatty acids (palmitic, oleic, and linoleic acids) in different ratios: a palmitate-enriched mixture (4:3:3) and an unsaturated fat-dominant mixture (2:4:4). In primary human monocyte-derived macrophages, palmitate enrichment increased CD14+CD11b+HLA-DR+ pro-inflammatory polarization, whereas the unsaturated fat-dominant mixture increased CD14+CD11b+CD163+ anti-inflammatory polarization. Mechanistic studies in THP-1-derived macrophages recapitulated these phenotype shifts and identified a reciprocal nuclear-receptor program: palmitate enrichment induced peroxisome proliferator-activated receptor gamma (PPARγ), together with ER-stress mediators EIF2AK3 and DDIT3, while the unsaturated fat-dominant mixture preferentially induced PPARα and IRF4. Pharmacologic modulation demonstrated functional dependence on PPARγ: GW9662 attenuated palmitate-driven M1-like polarization, whereas rosiglitazone disrupted the protective program under unsaturated fat-dominant conditions. These findings show that fatty acid composition, at equivalent total lipid concentration, is a dominant determinant of human macrophage inflammatory fate and highlight PPARγ as a context-dependent lipid sensor.

Keywords: PPARs; fatty acid; high fat diet; inflammation; macrophages.

Figure 1

Palmitic acid-rich diet reduces intracellular lipid accumulation and alters macrophage morphology. THP-1-derived macrophages were stimulated for 24 h with defined lipid mixtures to mimic dietary fatty acid environments: a palmitic acid-enriched formulation (HFD; PA:OA:LA = 4:3:3) or an unsaturated fat-dominant mixture (GFD; PA:OA:LA = 2:4:4). (A) Representative confocal micrographs showing macrophages stained with phalloidin (red, F-actin cytoskeleton), BODIPY 493/503 (green, neutral lipids), and DAPI (blue, nuclei). Scale bars = 100 µm. HFD treatment markedly reduced intracellular lipid droplet accumulation compared to GFD, as seen by lower BODIPY signal intensity. (B) Quantification of intracellular lipid content based on BODIPY fluorescence intensity normalized to cell count. Data represent mean ± SEM from n = 3 independent experiments. (C) Flow cytometric analyses showing changes in cell complexity (side scatter, SSC) and size (forward scatter, FSC). Representative dot plots display increased granularity in HFD-treated macrophages, consistent with morphological activation. Along with quantification bar graph of granular (high-SSC) macrophage populations expressed as percentage of total cells. Data are presented as mean ± SEM (n = 3); * p < 0.05, ** p < 0.01 by one-way ANOVA with Tukey’s post hoc test.

Figure 2

Fatty acid composition dictates pro- and anti-inflammatory macrophage polarization in human and THP-1-derived macrophages. To assess how defined fatty acid ratios regulate macrophage polarization, primary human monocyte-derived macrophages and THP-1-derived macrophages were stimulated for 24 h with vehicle (Control), a palmitate-enriched lipid mixture (HFD; PA:OA:LA = 4:3:3), or an unsaturated fat-dominant mixture (GFD; PA:OA:LA = 2:4:4). (A) Representative flow cytometry plots and quantification showing increased frequencies of CD14⁺CD11b⁺HLA-DR⁺ macrophages in primary human macrophages exposed to the palmitate-enriched mixture, consistent with M1-like polarization. (B) Representative plots and quantification demonstrating increased CD14⁺CD11b⁺CD163⁺ macrophages under unsaturated fat-dominant conditions, indicative of an M2-like phenotype in primary human macrophages. (C) Derived M1/M2 ratio in human macrophages, showing a significant shift toward pro-inflammatory polarization under palmitate-enriched conditions relative to GFD. To assess how distinct dietary fatty acid ratios modulate macrophage polarization in THP-1 model, THP-1-derived macrophages were stimulated in a similar manner. (D) Representative dot plots and quantification of CD11b⁺HLA-DR⁺ cells showing a significant increase in the M1-like population upon HFD treatment compared to control and GFD conditions. (E) Flow cytometric analysis and quantification of CD11b⁺CD163⁺ cells revealed that GFD treatment promoted an M2-like phenotype, whereas HFD exposure suppressed this anti-inflammatory subset. (F) Ratio of M1/M2 populations, demonstrating a pronounced shift toward M1 polarization in palmitate-enriched (HFD) conditions. (G,H) Representative immunoblot and densitometric quantification of CD11c (150 kDa) protein levels normalized to β-actin (42 kDa). GFD treatment reduced CD11c expression relative to HFD, further supporting an anti-inflammatory shift. Data are presented as mean ± SEM (n = 3 biological replicates, with each replicate representing an independent human donor.); * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 by one-way ANOVA with Tukey’s post hoc test.

Figure 3

Differential regulation of PPARγ and PPARα defines the divergent effects of HFD and GFD on macrophage transcriptional programming. To delineate the molecular mechanisms driving lipid ratio-dependent macrophage polarization, primary human monocyte-derived macrophages and THP-1-derived macrophages were stimulated with either palmitate-enriched HFD (PA:OA:LA = 4:3:3) or unsaturated fat-dominant GFD (PA:OA:LA = 2:4:4) for 24 h, followed by transcriptional analysis of nuclear receptors and stress-responsive regulators. (A–E) Quantitative PCR analysis revealed selective induction of PPAR isoforms under HFD vs. GFD conditions. (E–H) The ER stress sensor DDIT3 (CHOP) and its upstream effector EIF2AK3 (PERK) were significantly upregulated in response to HFD, highlighting activation of the unfolded protein response under palmitate-rich conditions. (I,J) Expression of sterol regulatory element-binding transcription factors showed that SREBF1 was downregulated in both treatment groups, consistent with reduced lipogenesis, whereas SREBF2 was preferentially elevated in GFD-treated cells, suggesting enhanced cholesterol remodeling in response to unsaturated fatty acids. (K–M) The cytokine suppressor SOCS3 was increased under both lipid conditions, while IRF4 expression was modestly increased under unsaturated fat-dominant conditions, consistent with an associative rather than causative role in macrophage polarization. (N–P) Expression of IRF5, ISG15, and STAT3 remained unaltered, indicating selective transcriptional reprogramming rather than a global activation response. Data are presented as mean ± SEM (n = 3 biological replicates); * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 by one-way ANOVA with Tukey’s post hoc test.

Figure 4

PPARγ activity is necessary and sufficient for lipid ratio-dependent macrophage polarization. To validate PPARγ expression at the protein level we conducted the following: (A) Representative immunoblot and densitometric quantification of PPARγ (50–60 kDa) protein levels normalized to β-actin (42 kDa). To functionally validate the role of PPARγ in mediating lipid ratio-driven macrophage polarization, THP-1-derived macrophages were treated with either the PPARγ antagonist GW9662 or the PPARγ agonist rosiglitazone under palmitate-enriched (HFD; PA:OA:LA = 4:3:3) or unsaturated fat-dominant (GFD; PA:OA:LA = 2:4:4) conditions for 24 h. (B) Representative confocal micrographs showing F-actin cytoskeleton (phalloidin, red), lipid droplets (BODIPY 493/503, green), and nuclei (DAPI, blue). HFD-treated macrophages exhibited reduced lipid accumulation and increased cell granularity compared to GFD, whereas pharmacologic inhibition of PPARγ with GW9662 restored intracellular lipid deposition and a rounded morphology under HFD conditions. Conversely, PPARγ activation with rosiglitazone in GFD-treated cells markedly reduced lipid content and promoted an elongated, stress-associated morphology. Insets highlight characteristic morphological features. Scale bars = 1 mm. (C,D) Quantitative analysis of lipid accumulation (BODIPY intensity per cell) and morphological parameters (granularity and circularity) confirmed that PPARγ inhibition reverses, while PPARγ activation enhances, lipid ratio-dependent phenotypic remodeling. Data represent mean ± SEM from three independent experiments; *** p < 0.001 and **** p < 0.0001 by one-way ANOVA with Tukey’s post hoc test.

Figure 5

PPARγ modulation alters lipid-ratio-driven macrophage polarization in primary human monocyte-derived macrophages. Primary human macrophages isolated from PBMCs were treated with a palmitate-enriched lipid mixture (HFD; PA: OA:LA = 4:3:3) or an unsaturated fat-dominant mixture (GFD; PA:OA:LA = 2:4:4) for 24 h in the presence or absence of the PPARγ antagonist GW9662 or the PPARγ agonist rosiglitazone. (A,B) Representative flow cytometry plots showing CD14+CD11b⁺HLA-DR⁺ (M1-like) and CD14+CD11b⁺CD163⁺ (M2-like) subsets under each condition. (C) Quantification of M1, M2, and M1/M2 ratios. Data represent mean ± SEM (n = 3 biological replicates, with each replicate representing an independent human donor.); **** p < 0.0001 by one-way ANOVA with Tukey’s post hoc test.