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Docosahexanoic acid antagonizes TNF-α-induced necroptosis by attenuating oxidative stress, ceramide production, lysosomal dysfunction, and autophagic features

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Abstract

Objective

It was previously reported that docosahexanoic acid (DHA) reduces TNF-α-induced necrosis in L929 cells. However, the mechanisms underlying this reduction have not been investigated. The present study was designed to investigate cellular and biochemical mechanisms underlying the attenuation of TNF-α-induced necroptosis by DHA in L929 cells.

Methods

L929 cells were pre-treated with DHA prior to exposure to TNF-α, zVAD, or Necrostatin-1 (Nec-1). Cell death and survival were assessed by MTT and caspase activity assays, and microscopic visualization. Reactive oxygen species (ROS) were measured by flow cytometry. C16- and C18-ceramides were measured by mass spectrometry. Lysosomal membrane permeabilization (LMP) was evaluated by fluorescence microscopy and flow cytometry using Acridine Orange. Cathepsin L activation was evaluated by immunoblotting and fluorescence microscopy. Autophagy was assessed by immunoblotting of LC3-II and Beclin.

Results

Exposure of L929 cells to TNF-α alone for 24 h induced necroptosis, as evidenced by the inhibition of cell death by Nec-1, absence of caspase-3 activity and Lamin B cleavage, and morphological analysis. DHA attenuated multiple biochemical events associated with TNF-α-induced necroptosis, including ROS generation, ceramide production, lysosomal dysfunction, cathepsin L activation, and autophagic features. DHA also attenuated zVAD-induced necroptosis but did not attenuate the enhanced apoptosis and necrosis induced by the combination of TNF-α with Actinomycin D or zVAD, respectively, suggesting that its protective effects might be limited by the strength of the cell death insult induced by TNF-α.

Conclusions

DHA effectively attenuates TNF-α-induced necroptosis and autophagy, most likely via its ability to inhibit TNF-α-induced sphingolipid metabolism and oxidative stress. These results highlight the role of this Omega-3 fatty acid in antagonizing inflammatory cell death.

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Acknowledgments

We are grateful to Leanna Ursales, Teka-Ann Haynes, Anamika Basu, Tino Sanchez, and other members of the Casiano and De Leon laboratories for their technical and editorial assistance. This work was supported by NIH grants 5P20MD006988 (MDL and CAC), 5P20MD001632 (MDL and CAC), and 5R25GM060507 (MDL).

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Authors and Affiliations

  1. Center for Health Disparities and Molecular Medicine and Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA, 92350, USA

    Fabio J. Pacheco, Frankis G. Almaguel, Whitney Evans, Leslimar Rios-Colon, Valery Filippov, Lai S. Leoh, Elizabeth Rook-Arena, Melanie Mediavilla-Varela, Marino De Leon & Carlos A. Casiano

  2. Faculty of Medicine, School of Health Sciences, River Plate Adventist University, Libertador San Martin, Puiggari, Entre Rios, Argentina

    Fabio J. Pacheco

  3. Division of Rheumatology, Department of Medicine, Loma Linda University School of Medicine, Loma Linda, CA, USA

    Elizabeth Rook-Arena & Carlos A. Casiano

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  1. Fabio J. Pacheco
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  2. Frankis G. Almaguel
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  3. Whitney Evans
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  4. Leslimar Rios-Colon
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  6. Lai S. Leoh
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  8. Melanie Mediavilla-Varela
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  9. Marino De Leon
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  10. Carlos A. Casiano
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Correspondence to Carlos A. Casiano.

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Responsible Editor: Graham R. Wallace.

M. D. Leon, and C. A. Casiano: these authors co-supervised this work and contributed equally to the underlying ideas and experimental design, and share senior authorship.

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Pacheco, F.J., Almaguel, F.G., Evans, W. et al. Docosahexanoic acid antagonizes TNF-α-induced necroptosis by attenuating oxidative stress, ceramide production, lysosomal dysfunction, and autophagic features. Inflamm. Res. 63, 859–871 (2014). https://doi.org/10.1007/s00011-014-0760-2

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