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. 2013 Sep 30;8(9):e76233.
doi: 10.1371/journal.pone.0076233. eCollection 2013.

Lysophosphatidylcholine triggers TLR2- and TLR4-mediated signaling pathways but counteracts LPS-induced NO synthesis in peritoneal macrophages by inhibiting NF-κB translocation and MAPK/ERK phosphorylation

Alan Brito Carneiro  1 , Bruna Maria Ferreira Iaciura, Lilian Lie Nohara, Carla Duque Lopes, Esteban Mauricio Cordero Veas, Vania Sammartino Mariano, Patricia Torres Bozza, Ulisses Gazos Lopes, Georgia Correa Atella, Igor Correia Almeida, Mário Alberto Cardoso Silva-Neto
Affiliations

Lysophosphatidylcholine triggers TLR2- and TLR4-mediated signaling pathways but counteracts LPS-induced NO synthesis in peritoneal macrophages by inhibiting NF-κB translocation and MAPK/ERK phosphorylation

Alan Brito Carneiro et al. PLoS One. .

Abstract

Background: Lysophosphatidylcholine (LPC) is the main phospholipid component of oxidized low-density lipoprotein (oxLDL) and is usually noted as a marker of several human diseases, such as atherosclerosis, cancer and diabetes. Some studies suggest that oxLDL modulates Toll-like receptor (TLR) signaling. However, effector molecules that are present in oxLDL particles and can trigger TLR signaling are not yet clear. LPC was previously described as an attenuator of sepsis and as an immune suppressor. In the present study, we have evaluated the role of LPC as a dual modulator of the TLR-mediated signaling pathway.

Methodology/principal findings: HEK 293A cells were transfected with TLR expression constructs and stimulated with LPC molecules with different fatty acid chain lengths and saturation levels. All LPC molecules activated both TLR4 and TLR2-1 signaling, as evaluated by NF-қB activation and IL-8 production. These data were confirmed by Western blot analysis of NF-қB translocation in isolated nuclei of peritoneal murine macrophages. However, LPC counteracted the TLR4 signaling induced by LPS. In this case, NF-қB translocation, nitric oxide (NO) synthesis and the expression of inducible nitric oxide synthase (iNOS) were blocked. Moreover, LPC activated the MAP Kinases p38 and JNK, but not ERK, in murine macrophages. Interestingly, LPC blocked LPS-induced ERK activation in peritoneal macrophages but not in TLR-transfected cells.

Conclusions/significance: The above results indicate that LPC is a dual-activity ligand molecule. It is able to trigger a classical proinflammatory phenotype by activating TLR4- and TLR2-1-mediated signaling. However, in the presence of classical TLR ligands, LPC counteracts some of the TLR-mediated intracellular responses, ultimately inducing an anti-inflammatory phenotype; LPC may thus play a role in the regulation of cell immune responses and disease progression.

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Conflict of interest statement

Competing Interests: Dr. Patricia T. Bozza is a co-author in this study and she is also a member of the PLOS ONE Editorial Board. This condition does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. LPC triggers NF-қB activation through either TLR4- or TLR2/1-dependent signaling pathways.
HEK 293A cells were transfected in three different groups. Groups A and B received expression constructs for TLR4 (A) or TLR2 and TLR1 (B). Both also received MD-2, CD14, and CD36 constructs and the ELAM-1-firefly luciferase and β-actin-Renilla luciferase reporter plasmids. The third group (C) received only the empty vector pDisplay and the luciferase reporter plasmids. Groups A and B were separately stimulated with 0.1, 1, 10, 100 and 200 µM of different types of LPC (Sigma; C14:0, C16:0, C18:0, and C18:1), 100 ng/mL of LPS and 1 nM of Pam3CSK4 (P3C). Group C was stimulated with LPS, Pam3Cys or 0.1, 1, 10 and 100 µM of LPC (C16:0). The agonists were diluted in DMEM medium with 10% bovine fetal serum. After 4 h of incubation, luciferase activity was measured and expressed as the ratio of NF-қB-dependent firefly luciferase activity to the control Renilla luciferase activity. Data is the mean ± S.E. of two different experiments. ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).
Figure 2
Figure 2. LPC triggers IL-8 production through either TLR4- or TLR2/1-dependent signaling pathways.
HEK 293A cells were transfected and stimulated as described on Figure 1. After 20 hours of incubation, IL-8 production was measured by the ELISA assay. Data is the mean ± S.E. of two different experiments. ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).
Figure 3
Figure 3. LPC inhibits NF-қB translocation, iNOS expression, and NO production in LPS-stimulated macrophages.
Peritoneal macrophages from BALB/cmice were incubated in the absence or presence of 1 µg/mL LPS and different concentrations of LPC (Sigma) at 37 °C in a 5% CO2 atmosphere. After 1 h of incubation, NF-қB translocation (A) was assayed by Western blot analysis. After 24 hours, NO production (B) was assayed by measuring the amount of nitrite in the culture supernatant using the Griess reagent, and iNOS expression (C) was determined by Western blot analysis followed by densitometry (lower panel). Data is the mean ± S.D. of three different experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).
Figure 4
Figure 4. LPC activates JNK and p38, but not ERK, in macrophages.
Peritoneal macrophages from BALB/c mice were incubated in the absence or presence of different concentrations of LPC mix (Sigma) for 20 min at 37 °C in a 5% CO2 atmosphere, and the cytoplasm content was homogenized and assayed as follows. The Phospho-MAPK array was used for analysis of enzymatic activation (A). The reaction was visualized with the enhanced chemiluminescent system and subjected to densitometric analysis (***, p< 0.001, ANOVA). Protein levels of the phosphorylated MAPKs JNK (B), p38 (C) and ERK (D) were determined by Western blot. Data is the mean ± S.E. of two different experiments.
Figure 5
Figure 5. LPC inhibits LPS-induced ERK activation.
Peritoneal macrophages from BALB/c mice were incubated in the absence or presence of 1 µg/mL LPS or in the presence or absence of the indicated concentrations of LPC (Sigma) at 37 °C in a 5% CO2 atmosphere (A, D, E). In parallel HEK 293A cells with TLR constructs as indicated (B, C). Each group received expression constructs for TLR4 (B) or both TLR2 and TLR1 (C), as well as MD-2, CD14 and CD36 plasmids. The cells were then incubated in the absence or presence of 100 ng/mL LPS or 1 nM Pam3CSK4 (P3C) and 10 or 100 µM of LPC, for 40 min at 37 °C in a 5% CO2 atmosphere. After incubation either macrophages or HEK cells were homogenized, the protein levels was determined and samples evaluated by Western blot with the use of antibodies against p-ERK (A, B, C), p-JNK (D) and p-P38 (E). Loading controls were run with the use of antibodies raised towards actin. Experiments were performed at least two times with different animals and samples.
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