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Cited by 216Inflammasome and immune disordersFatty Acid Research and HealthImmune Response and Inflammation

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Omega-3 Free Fatty Acids Suppress Macrophage Inflammasome Activation by Inhibiting NF-κB Activation and Enhancing Autophagy

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Williams-Bey, Yolanda, Boularan, Cedric, Vural, Ali, Huang, Ning-Na, Hwang, Il-Young, Shan-Shi, Chong, Kehrl, John H.·2014·PLoS ONE
DOI 10.1371/journal.pone.0097957PMC PMC4049592
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Abstract

The omega-3 (ω3) fatty acid docosahexaenoic acid (DHA) can suppress inflammation, specifically IL-1β production through poorly understood molecular mechanisms. Here, we show that DHA reduces macrophage IL-1β production by limiting inflammasome activation. Exposure to DHA reduced IL-1β production by ligands that stimulate the NLRP3, AIM2, and NAIP5/NLRC4 inflammasomes. The inhibition required Free Fatty Acid Receptor (FFAR) 4 (also known as GPR120), a G-protein coupled receptor (GPR) known to bind DHA. The exposure of cells to DHA recruited the adapter protein β-arrestin1/2 to FFAR4, but not to a related lipid receptor. DHA treatment reduced the initial inflammasome priming step by suppressing the nuclear translocation of NF-κB. DHA also reduced IL-1β levels by enhancing autophagy in the cells. As a consequence macrophages derived from mice lacking the essential autophagy protein ATG7 were partially resistant to suppressive effects of DHA. Thus, DHA suppresses inflammasome activation by two distinct mechanisms, inhibiting the initial priming step and by augmenting autophagy, which limits inflammasome activity.

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Figure 1 DHA suppresses inflammasome induced IL-1β production. LPS primed. (A) THP-1 cells and (B) BMDMs were stimulated with ATP. DHA (50 uM or 100 uM) added or not at the priming step. Supernatants were collected and an ELISA was performed to check IL-1β levels. (C) Western blot analysis of cell supernatants and lysates from LPS primed THP-1 cells treated with ATP in the presence or absence of DHA. The indicated samples were analyzed for mature IL-1β, pro- and mature caspase-1, ASC, NLRP3, and actin. (D) BMDMs were LPS primed and stimulated with the inflammasome inducers nigericin, Poly(dA:dT), and flagellin. DHA was added, or not, at the priming step. Supernatants were collected and IL-1β levels checked by ELISA. Results are representatives of a minimum of three independent experiments. * P<0.05, **P<0.001, ***P<0.0002, ****P<0.0001.
Figure 1 DHA suppresses inflammasome induced IL-1β production. LPS primed. (A) THP-1 cells and (B) BMDMs were stimulated with ATP. DHA (50 uM or 100 uM) added or not at the priming step. Supernatants were collected and an ELISA was performed to check IL-1β levels. (C) Western blot analysis of cell supernatants and lysates from LPS primed THP-1 cells treated with ATP in the presence or absence of DHA. The indicated samples were analyzed for mature IL-1β, pro- and mature caspase-1, ASC, NLRP3, and actin. (D) BMDMs were LPS primed and stimulated with the inflammasome inducers nigericin, Poly(dA:dT), and flagellin. DHA was added, or not, at the priming step. Supernatants were collected and IL-1β levels checked by ELISA. Results are representatives of a minimum of three independent experiments. * P<0.05, **P<0.001, ***P<0.0002, ****P<0.0001.CC BY · PubMed Central
Figure 2 DHA inhibits the translocation of NF-κB to the nucleus. FFAR4-GFP expressing LPS primed non-differentiated THP-1 cells were stimulated with ATP or nigericin. DHA (100 µM) was added or not at the priming step. PFA fixed cells were permeablized and then stained with p65 antibody and analysis was performed using an ImageStream instrument. Shown are the (A) Imaging results and (B) Flow cytometry results. (C) ImageStream analysis of nuclear p65 NF-κB in non-differentiated THP-1 cells expressing FFAR1-GFP or FFAR4-GFP and exposed to LPS, or not, in the presence of DHA (50 µM), or not. (D) Confocal microscopy of BMDMs LPS primed and treated with nigericin in the presence, or absence of DHA, to examine the status of NF-κB translocation by p65 immunostaining. Shown are representative individual images. Scale bar is 20 µM. Whisker plot shows the amount of nuclear p65 immunofluorescence in the nuclei of BMDMs treated as indicated. Data are representative of three independent experiments. **P<0.001, n.s-not significant.
Figure 2 DHA inhibits the translocation of NF-κB to the nucleus. FFAR4-GFP expressing LPS primed non-differentiated THP-1 cells were stimulated with ATP or nigericin. DHA (100 µM) was added or not at the priming step. PFA fixed cells were permeablized and then stained with p65 antibody and analysis was performed using an ImageStream instrument. Shown are the (A) Imaging results and (B) Flow cytometry results. (C) ImageStream analysis of nuclear p65 NF-κB in non-differentiated THP-1 cells expressing FFAR1-GFP or FFAR4-GFP and exposed to LPS, or not, in the presence of DHA (50 µM), or not. (D) Confocal microscopy of BMDMs LPS primed and treated with nigericin in the presence, or absence of DHA, to examine the status of NF-κB translocation by p65 immunostaining. Shown are representative individual images. Scale bar is 20 µM. Whisker plot shows the amount of nuclear p65 immunofluorescence in the nuclei of BMDMs treated as indicated. Data are representative of three independent experiments. **P<0.001, n.s-not significant.CC BY · PubMed Central
Figure 3 DHA-mediated suppression of inflammasome activity depends upon FFAR4. (A) Quantitative RT-PCR to detect Ffar1, Gpr84, and Ffar4 mRNA expression in BMDMs. Cells were LPS primed and ATP treated. DHA (100 µM) added during priming step, or not. Data is relative to Gapdh expression x 105. Experiment performed 2 times with similar results. (B) Quantitative RT-PCR to detect GPR84 or FFAR4 mRNA in LPS primed THP-1 cells expressing siRNAs targeting FFAR4 or GPR84, respectively. The results were normalized to β-actin expression and shown as relative to control. (C) IL-1β ELISA to assess inflammasome activity in FFAR1, GPR84, or FFAR4 knock-down cells. LPS primed THP-1 cells expressing siRNAs targeting FFAR1, GPR84, or FFAR4 were stimulated with ATP. DHA added during the priming step. Similar results from 3 experiments. *P<0.05, **P<0.001, ****P<0.0001.
Figure 3 DHA-mediated suppression of inflammasome activity depends upon FFAR4. (A) Quantitative RT-PCR to detect Ffar1, Gpr84, and Ffar4 mRNA expression in BMDMs. Cells were LPS primed and ATP treated. DHA (100 µM) added during priming step, or not. Data is relative to Gapdh expression x 105. Experiment performed 2 times with similar results. (B) Quantitative RT-PCR to detect GPR84 or FFAR4 mRNA in LPS primed THP-1 cells expressing siRNAs targeting FFAR4 or GPR84, respectively. The results were normalized to β-actin expression and shown as relative to control. (C) IL-1β ELISA to assess inflammasome activity in FFAR1, GPR84, or FFAR4 knock-down cells. LPS primed THP-1 cells expressing siRNAs targeting FFAR1, GPR84, or FFAR4 were stimulated with ATP. DHA added during the priming step. Similar results from 3 experiments. *P<0.05, **P<0.001, ****P<0.0001.CC BY · PubMed Central
Figure 4 DHA elicits a calcium flux and its suppression of inflammasome activity partially depends upon β-arrestin2 not β-arrestin1. (A) DHA elicited changes in intracellular Ca2+ in BMDMs treated with pertussis toxin (200 ng/ml), or not, for 2 hours prior to the assay. (B) BRET analysis using HeLa cells expressing a BRET donor (RLuc-β-arrestin1 or β-arrestin2-RLuc) and increasing amounts of a BRET acceptor (FFAR4-GFP or FFAR1-GFP) stimulated with LPS, or not, prior to addition of DHA. The BRET ratios were calculated and the change in BRET determined. (C) Confocal microscopy of HeLa cells expressing FFAR4-GFP and β-arrestin-2 mCherry following exposure to DHA. Red arrow indicates β-arrestin-2 recruitment and the white arrows intracellular co-localization of β-arrestin-2 and FFAR4. Scale bar is 10 µM. (D) Intracellular calcium response to DHA of BMDMs prepared from wild type and β-arrestin knock-out mice. (E) ELISA to measure IL-1β in cell supernatants conditioned by LPS primed and ATP treated BMDMs from WT, arrb1 −/−, and arrb2 −/− mice. DHA (100 M) was added at the priming step. Results are from 3 independent experiments. * P<0.05, ****P<0.0001.
Figure 4 DHA elicits a calcium flux and its suppression of inflammasome activity partially depends upon β-arrestin2 not β-arrestin1. (A) DHA elicited changes in intracellular Ca2+ in BMDMs treated with pertussis toxin (200 ng/ml), or not, for 2 hours prior to the assay. (B) BRET analysis using HeLa cells expressing a BRET donor (RLuc-β-arrestin1 or β-arrestin2-RLuc) and increasing amounts of a BRET acceptor (FFAR4-GFP or FFAR1-GFP) stimulated with LPS, or not, prior to addition of DHA. The BRET ratios were calculated and the change in BRET determined. (C) Confocal microscopy of HeLa cells expressing FFAR4-GFP and β-arrestin-2 mCherry following exposure to DHA. Red arrow indicates β-arrestin-2 recruitment and the white arrows intracellular co-localization of β-arrestin-2 and FFAR4. Scale bar is 10 µM. (D) Intracellular calcium response to DHA of BMDMs prepared from wild type and β-arrestin knock-out mice. (E) ELISA to measure IL-1β in cell supernatants conditioned by LPS primed and ATP treated BMDMs from WT, arrb1 −/−, and arrb2 −/− mice. DHA (100 M) was added at the priming step. Results are from 3 independent experiments. * P<0.05, ****P<0.0001.CC BY · PubMed Central
Figure 5 DHA regulates inflammasome activity by promoting autophagy. (A) Confocal microscopy of BMDMs from GFP-LC3 mice primed with LPS and treated with nigericin. DHA (50 µM) was added at the priming step. Scale bar is 5 µM. (B) Number of cells with >5 GFP-LC3 puncta per cell were determined for each treatment group. Data are from 3 independent experiments. (C) ELISA to measure IL-1β in supernatants conditioned by BMDMs prepared from Atg7 flox/flox or Atg7 flox/flox Vav1-Cre mice. The cells were primed with LPS and treated with ATP. DHA was added at the priming step. Results are from 3 independent experiments. (D) Western blot analysis of supernatants and cell lysates from BMDMs prepared from Atg7 flox/flox or Atg7 flox/flox Vav1-Cre mice. Cells were LPS primed and treated with ATP. DHA was added at the time of priming. The numbers indicate the relative expression of IL-1β in the cell supernatants. **P<0.001, ***P<0.0002, ****P<0.0001. (E) DHA and ATP triggered changes in intracellular Ca+2 in mouse BMDMs. The cells were exposed to the indicated concentrations of DHA and/or ATP. Results from one experiment performed in triplicate. Representative of three experiments performed.
Figure 5 DHA regulates inflammasome activity by promoting autophagy. (A) Confocal microscopy of BMDMs from GFP-LC3 mice primed with LPS and treated with nigericin. DHA (50 µM) was added at the priming step. Scale bar is 5 µM. (B) Number of cells with >5 GFP-LC3 puncta per cell were determined for each treatment group. Data are from 3 independent experiments. (C) ELISA to measure IL-1β in supernatants conditioned by BMDMs prepared from Atg7 flox/flox or Atg7 flox/flox Vav1-Cre mice. The cells were primed with LPS and treated with ATP. DHA was added at the priming step. Results are from 3 independent experiments. (D) Western blot analysis of supernatants and cell lysates from BMDMs prepared from Atg7 flox/flox or Atg7 flox/flox Vav1-Cre mice. Cells were LPS primed and treated with ATP. DHA was added at the time of priming. The numbers indicate the relative expression of IL-1β in the cell supernatants. **P<0.001, ***P<0.0002, ****P<0.0001. (E) DHA and ATP triggered changes in intracellular Ca+2 in mouse BMDMs. The cells were exposed to the indicated concentrations of DHA and/or ATP. Results from one experiment performed in triplicate. Representative of three experiments performed.CC BY · PubMed Central

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Metadata

Authors
Williams-Bey, Y., Boularan, C., Vural, A., Huang, N.-N., Hwang, I.-Y., Shan-Shi, C., & Kehrl, J. H.
Affiliations
National Institutes of Health (US)
Corresponding author
Kehrl, John H.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2014-06-09
Volume
9
Issue
6
Pages
e97957
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0097957
PMC
PMC4049592
OpenAlex
W2062456565
Cited by
215
References
18
Citations by year
2026: 15 · 2025: 12 · 2024: 10 · 2023: 11 · 2022: 19 · 2021: 28 · 2020: 23 · 2019: 13 · 2018: 19 · 2017: 19 · 2016: 21 · 2015: 23 · 2014: 2
FWCI
8.88
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Institutes of Health
  • National Institute of Allergy and Infectious Diseases
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Inflammasome and immune disorders
  • Health Sciences › Nursing › Nutrition and Dietetics › Fatty Acid Research and Health
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
Keywords
docosahexaenoic acid, omega-3 fatty acids, macrophages, inflammasome activation, IL-1β production, NLRP3 inflammasome, AIM2 inflammasome, free fatty acid receptor 4, GPR120, NF-κB nuclear translocation, autophagy, ATG7
MeSH terms
beta-Arrestins, Animals, Autophagy, Bone Marrow Cells, Calcium, Cell Nucleus, Docosahexaenoic Acids, Gene Expression Regulation, HeLa Cells, Humans, Lipopolysaccharides, Macrophage Activation, Macrophages, Mice, Inbred C57BL, NF-kappa B, Arrestins, Active Transport, Cell Nucleus, Intracellular Space, Receptors, G-Protein-Coupled, Mice, Interleukin-1beta, Inflammasomes

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