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Saturated fatty acids activate TLR-mediated proinflammatory signaling pathways.

ABSTRACT: Toll-like receptor 4 (TLR4) and TLR2 were shown to be activated by saturated fatty acids (SFAs) but inhibited by docosahexaenoic acid (DHA). However, one report suggested that SFA-induced TLR activation in cell culture systems is due to contaminants in BSA used for solubilizing fatty acids. This report raised doubt about proinflammatory effects of SFAs. Our studies herein demonstrate that sodium palmitate (C16:0) or laurate (C12:0) without BSA solubilization induced phosphorylation of inhibitor of nuclear factor-?B ?, c-Jun N-terminal kinase (JNK), p44/42 mitogen-activated-kinase (ERK), and nuclear factor-?B subunit p65, and TLR target gene expression in THP1 monocytes or RAW264.7 macrophages, respectively, when cultured in low FBS (0.25%) medium. C12:0 induced NF?B activation through TLR2 dimerized with TLR1 or TLR6, and through TLR4. Because BSA was not used in these experiments, contaminants in BSA have no relevance. Unlike in suspension cells (THP-1), BSA-solubilized C16:0 instead of sodium C16:0 is required to induce TLR target gene expression in adherent cells (RAW264.7). C16:0-BSA transactivated TLR2 dimerized with TLR1 or TLR6 and through TLR4 as seen with C12:0. These results and additional studies with the LPS sequester polymixin B and in MyD88(-/-) macrophages indicated that SFA-induced activation of TLR2 or TLR4 is a fatty acid-specific effect, but not due to contaminants in BSA or fatty acid preparations.

SUBMITTER: Huang S

PROVIDER: S-EPMC3413240 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Saturated fatty acids activate TLR-mediated proinflammatory signaling pathways.

Huang Shurong S Rutkowsky Jennifer M JM Snodgrass Ryan G RG Ono-Moore Kikumi D KD Schneider Dina A DA Newman John W JW Adams Sean H SH Hwang Daniel H DH

Journal of lipid research 20120704 9

Toll-like receptor 4 (TLR4) and TLR2 were shown to be activated by saturated fatty acids (SFAs) but inhibited by docosahexaenoic acid (DHA). However, one report suggested that SFA-induced TLR activation in cell culture systems is due to contaminants in BSA used for solubilizing fatty acids. This report raised doubt about proinflammatory effects of SFAs. Our studies herein demonstrate that sodium palmitate (C16:0) or laurate (C12:0) without BSA solubilization induced phosphorylation of inhibitor ...[more]

PMID: 22766885

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Project description:Study questionWhat are the effects of fatty acids on placental inflammatory cytokine with respect to toll-like receptor-4/nuclear factor-kappa B (TLR4/NF-kB)?Summary answerExogenous fatty acids induce a pro-inflammatory cytokine response in human placental cells in vitro via activation of TLR4 signaling pathways.What is known alreadyThe placenta is exposed to changes in circulating maternal fatty acid concentrations throughout pregnancy. Fatty acids are master regulators of innate immune pathways through recruitment of toll-like receptors and activation of cytokine synthesis.Study design, size, durationTrophoblast cells isolated from 14 normal term human placentas were incubated with long chain fatty acids (FA) of different carbon length and degree of saturation. The expression and secretion of interleukin-6 (IL-6), IL-8 and tumor necrosis factor-alpha (TNF-α) were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Antibodies against TLR4 ligand binding domain, downstream signaling and anti-p65 NFkB-inhibitor were used to characterize the pathways of FA action.Participants/materials, setting, methodsGeneral approach used primary human term trophoblast cell culture. Methods and end-points used real-time quantitative PCR, cytokine measurements, immunohistochemistry, western blots.Main results and the role of chanceThe long chain saturated fatty acids, stearic and palmitic (PA), stimulated the synthesis as well as the release of TNF-α, IL-6 and IL-8 by trophoblast cells (2- to 6-fold, P < 0.001). In contrast, the unsaturated (palmitoleic, oleic, linoleic) acids did not modify cytokine expression significantly. Palmitate-induced inflammatory effects were mediated via TLR4 activation, NF-kB phosphorylation and nuclear translocation.Limitations, reasons for cautionTNF-α protein level was close to the limit of detection in the culture medium even when cells were cultured with PA.Wider implications of the findingsThese mechanisms open the way to a better understanding of how changes in maternal lipid homeostasis may regulate placental inflammatory status.Study funding/competing interestsX.Y. was recipient of fellowship award from West China Second University Hospital, Sichuan University (NIH HD 22965-19). The authors have nothing else to disclose.Trial registration numberNone.

Project description:IntroductionObesity during pregnancy increases the risk for maternal complications like gestational diabetes, preeclampsia, and maternal inflammation. Maternal obesity also increases the risk of childhood obesity, intrauterine growth restriction (IUGR) and diabetes to the offspring. Increased circulating free fatty acids (FFAs) in obesity due to adipose tissue lipolysis induces lipoapoptosis to hepatocytes, cholangiocytes, and pancreatic-β-cells. During the third trimester of human pregnancy, there is an increase in maternal lipolysis and release of FFAs into the circulation. It is currently unknown if increased FFAs during gestation as a result of maternal obesity cause placental cell lipoapoptosis. Increased exposure of FFAs during maternal obesity has been shown to result in placental lipotoxicity. The objective of the present study is to determine saturated FFA-induced trophoblast lipoapoptosis and also to test the protective role of monounsaturated fatty acids against FFA-induced trophoblast lipoapoptosis using in vitro cell culture model. Here, we hypothesize that saturated FFAs induce placental trophoblast lipoapoptosis, which was prevented by monounsaturated fatty acids.MethodsBiochemical and structural markers of apoptosis by characteristic nuclear morphological changes with DAPI staining, and caspase 3/7 activity was assessed. Cleaved PARP and cleaved caspase 3 were examined by western blot analysis.ResultsTreatment of trophoblast cell lines, JEG-3 and JAR cells with palmitate (PA) or stearate (SA) induces trophoblast lipoapoptosis as evidenced by a significant increase in apoptotic nuclear morphological changes and caspase 3/7 activity. We observed that saturated FFAs caused a concentration-dependent increase in placental trophoblast lipoapoptosis. We also observed that monounsaturated fatty acids like palmitoleate and oleate mitigates placental trophoblast lipoapoptosis caused due to PA exposure.ConclusionWe show that saturated FFAs induce trophoblast lipoapoptosis. Co-treatment of monounsaturated fatty acids like palmitoleate and oleate protects against FFA-induced trophoblast lipoapoptosis.

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Saturated fatty acids activate TLR-mediated proinflammatory signaling pathways.

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Saturated fatty acids activate TLR-mediated proinflammatory signaling pathways.

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