The Synergy between Palmitate and TNF-α for CCL2 Production Is Dependent on the TRIF/IRF3 Pathway: Implications for Metabolic Inflammation

Abstract

The chemokine CCL2 (also known as MCP-1) is a key regulator of monocyte infiltration into adipose tissue, which plays a central role in the pathophysiology of obesity-associated inflammation and insulin resistance. It remains unclear how CCL2 production is upregulated in obese humans and rodents. Because elevated levels of the free fatty acid (FFA) palmitate and TNF-α have been reported in obesity, we studied whether these agents interact to trigger CCL2 production. Our data show that treatment of THP-1 and primary human monocytic cells with palmitate and TNF-α led to a marked increase in CCL2 production compared with either treatment alone. Mechanistically, we found that cooperative production of CCL2 by palmitate and TNF-α did not require MyD88, but it was attenuated by blocking TLR4 or TRIF. IRF3-deficient cells did not show synergistic CCL2 production in response to palmitate/TNF-α. Moreover, IRF3 activation by polyinosinic-polycytidylic acid augmented TNF-α-induced CCL2 secretion. Interestingly, elevated NF-κB/AP-1 activity resulting from palmitate/TNF-α costimulation was attenuated by TRIF/IRF3 inhibition. Diet-induced C57BL/6 obese mice with high FFAs levels showed a strong correlation between TNF-α and CCL2 in plasma and adipose tissue and, as expected, also showed increased adipose tissue macrophage accumulation compared with lean mice. Similar results were observed in the adipose tissue samples from obese humans. Overall, our findings support a model in which elevated FFAs in obesity create a milieu for TNF-α to trigger CCL2 production via the TLR4/TRIF/IRF3 signaling cascade, representing a potential contribution of FFAs to metabolic inflammation.

FIGURE 1.

Palmitate (PA) and TNF-α synergistically induce CCL2 in monocytic cells. (A) THP-1 cells were treated for 24 h with PA (200 μM), alone or in combination with TNF-α (10 ng/ml), before harvest. Total RNA was extracted, and Ccl2 mRNA was quantified by real-time PCR. Relative mRNA expression was expressed as fold change. (B) Secreted CCL2 protein in culture media was determined by ELISA. (C and D) Primary monocytes were isolated from PBMCs of healthy volunteers. Monocytes were incubated with PA and/or TNF-α for 24 h. Ccl2 mRNA (C) and secreted protein (D) were determined by real-time RT-PCR and ELISA. (E) THP-1 cells were immune-stained for confocal microscopy, as described in Materials and Methods. CCL2 expression is shown by green fluorescence (inset), whereas nuclei are stained blue with DAPI (original magnification ×40). (F) THP-1–derived macrophages were treated, as described above, and CCL2 protein expression was determined. (G and H) Migration assay was performed as described in Materials and Methods. Sample fluorescence (OD), expressed as RFU, was measured using excitation at 480 nm and emission at 520 nm. The number of cells migrating was calculated based on RFU values of test samples. (I) THP-1 cells were treated for 24 h with different FFAs (oleate [200 μm], myristate [50 μM] laurate [50 μm]), alone or in combination with TNF-α. Secreted CCL2 protein in culture media was determined by ELISA. (J) Monocytic cells were treated with the short-chain fatty acid butyrate (50 μM), alone or in combination with TNF-α. CCL2 protein was determined. All data are expressed as mean ± SEM (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus PA or TNF-α alone.

FIGURE 2.

Interference of TLR4 suppresses the synergistic or additive production of CCL2. (A and B) Monocytic cells were treated with 2 μg/ml neutralizing TLR4 mAb or isotype-matched control (IgA2) for 40 min. Ab-treated cells were stimulated with palmitate (PA) or BSA and incubated for 24 h. Ccl2 mRNA was quantified by real-time PCR, and secreted CCL2 protein was determined in the culture media by ELISA. (C) Monocytic cells were transfected with control or TLR4 siRNA. TLR4-deficient cells were treated with PA and BSA for 24 h. ELISA was done to measure secreted CCL2 protein. (D and E) Monocytic cells were treated with LPS (10 ng/ml), alone or in combination with TNF-α, for 24 h. CCL2 expression was determined at the mRNA (D) and protein (E) levels. (F and G) Monocytic cells were treated with PA, alone or in combination with TNF-α, for 24 h. (F) Cells were stained with TNF-αR1 (CD120a FITC) Ab, along with matched control Ab (IgGa FITC), and assessed by flow cytometry. (G) Data are presented as a bar graph of mean fluorescence intensity (MFI). (H and I) Monocytic cells were treated with IL-1β (10 ng/ml), PA (200 μM), or both for 24 h. Ccl2 mRNA (H) and protein (I) levels were determined. (J and K) THP1-XBlue cells (monocytic cells stably expressing a SEAP reporter inducible by NF-κB and AP-1) were treated with PA and LPS, alone or in combination with TNF-α, for 24 h. Culture media were collected. Cell culture media were assayed for SEAP reporter activity (degree of NF-κB/AP-1 activation). All data are expressed as mean ± SEM (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus PA or TNF-α alone.

FIGURE 3.

Synergistic induction of CCL2 by palmitate (PA)/TNF-α involves an MyD88-independent mechanism. (A and B) MyD88-knockout (KO) cells (THP1-XBlue-defMyD cells) were treated with PA (200 μm), 0.1% BSA (vehicle), or TNF-α (10 ng/ml), alone or in combination. Cells and culture media were collected after 24 h. CCL2 gene expression was determined by real-time PCR, and secreted CCL2 protein was determined in culture media by ELISA. (C) Cell culture media were also assayed for SEAP reporter activity representing the degree of NF-κB/AP-1 activation. All data are expressed as mean ± SEM (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus PA or TNF-α alone.

FIGURE 4.

Synergistic CCL2 production by palmitate (PA) and TNF-α requires TRIF. (A and B) Monocytic cells were treated with CPZ (an inhibitor of endocytosis; 10 μM) for 1 h and then treated as indicated for 24 h. Cells were used for the isolation of total RNA to assess Ccl2 gene expression by real-time RT-PCR. Cell culture supernatants were assayed for secreted CCL2 protein, as determined by ELISA. (C and D) Cells were treated with resveratrol (a TRIF inhibitor; 5 μM) for 30 min, followed by treatments as indicated. CCL2 expression was determined at the mRNA and protein levels. (E and F) Cells were incubated with TRIF inhibitory peptide (Pepinh-TRIF is a 30-aa peptide that blocks TRIF signaling by interfering with TLR–TRIF interaction) for 5 h and then treated for 24 h, as indicated. Pepinh-TRIF: RQIKIWFQNRRMKWKK-FCEEFQVPGRGELH-NH2 (5 μM), control: RQIKIWFQNRRMKWKK-SLHGRGDPMEAFII-NH2 (5 μM). CCL2 expression was determined at the mRNA and protein levels. (G and H) Cells were treated with CPZ and resveratrol, followed by treatments as indicated. Cell supernatants were collected and assayed for SEAP reporter activity as a measure of NF-κB/AP-1 activation. All data are expressed as mean ± SEM (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus PA or TNF-α alone.

FIGURE 5.

Synergistic production of CCL2 by palmitate (PA)/TNF-α requires IRF3. (A) Monocytic cells were transfected with control or IRF3 siRNA and incubated for 40 h. Real-time PCR was performed to measure IRF3 expression. (B and C) IRF3-deficient cells were treated as indicated, and Ccl2 mRNA and protein expression was determined. (D and E) Poly I:C (an activator of IRF3) increased TNF-α–induced CCL2. Cells were treated (via transfection) with poly I:C (5 μg) for 2 h and then incubated with BSA, PA, or TNF-α, or a combination of PA and TNF-α, for 24 h. CCL2 mRNA and protein expression was determined. (F) Western blot showing phosphorylation of IRF3 after PA or poly I:C treatment. (G) Cells were treated as indicated. Fixed and permeabilized cells were stained for nuclei and IRF3 using DAPI and anti-IRF3 Ab. Confocal laser scanning microscopy showed immunocytochemical localization of nuclei (blue; top panels), IRF3 (red; middle panels), and merged images of nuclei and IRF3 with magnified insets (bottom panels). All data are expressed as mean ± SEM (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus PA or TNF-α alone.

FIGURE 6.

Elevated CCL2 is correlated with TNF-α in DIO mice and obese humans. (A) Male mice were fed an HFD (n = 11) or chow (n = 9) for 16 wk. Plasma levels of TNF-α, CCL2, and FFAs were determined. (B) Correlation between plasma levels of CCL2 and TNF-α in obese mice. (C) WAT samples were obtained from DIO mice (n = 7). Adipose tissue CCL2 and TNF-α protein expression was determined by IHC. Staining intensity is shown in arbitrary units (AU). CCL2 protein expression correlated positively with TNF-α protein expression in the adipose tissue of obese mice. (D and E) Sections of s.c. adipose tissue from lean/OW (n = 26) and obese (n = 26) individuals. Increased adipose tissue expression of mRNA of Ccl2 and Tnf-α was detected by real-time RT-PCR and represented as fold change over controls. Each dot represents an individual value for Ccl2 or Tnf-α mRNA. Horizontal lines represent the mean values of adipose tissue CCL2 and TNF-α in each group. (F) Correlation of Ccl2 mRNA with Tnf-α mRNA in obese individuals. (G) CCL2 and TNF-α protein expression was detected by IHC. Protein expression was represented as staining intensity based on Aperio-positive pixel counts (Aperio software algorithm version 9.0). Correlation of CCL2 protein levels with TNF-α protein in obese individuals. *p < 0.05, **p < 0.01 versus controls.

FIGURE 7.

Schematic illustration of signaling pathways underlying the synergy between palmitate and TNF-α for CCL2 production. The pathway highlighted with red arrows represents the predominant mechanism of this synergistic response induced by palmitate. CPZ may inhibit TLR4 internalization and suppress synergy between palmitate/TNF-α for production of CCL2.