Email updates

Keep up to date with the latest news and content from Critical Care and BioMed Central.

This article is part of the supplement: Sepsis 2012

Poster presentation

LPS-induced Pellino3 degradation is mediated by p62-dependent autophagy

A Heeg1*, L Kuchler1, LK Eifler1, T Knape2, H Heide3, B Brüne1 and A von Knethen1

  • * Corresponding author: A Heeg

Author Affiliations

1 Institute of Biochemistry I - Pathobiochemistry, Goethe-University Frankfurt, Germany

2 Fraunhofer IME Project Group - Translational Medicine and Pharmacology, Frankfurt, Germany

3 Molecular Bioenergetics - Centre of Biological Chemistry, Goethe-University Frankfurt, Germany

For all author emails, please log on.

Critical Care 2012, 16(Suppl 3):P50  doi:10.1186/cc11737


The electronic version of this article is the complete one and can be found online at: http://ccforum.com/content/16/S3/P50


Published:14 November 2012

© 2012 Heeg et al.; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background

In macrophages Toll-like receptor 4 (TLR4) is activated in response to lipopolysaccharide (LPS) and induces proinflammatory cytokine expression [1]. Therefore, mechanisms terminating proinflammatory gene expression are important. Autophagy plays a central role in controlling innate immune responses by lysosomal degradation of signaling proteins, thus contributing to the resolution of inflammation [2]. Autophagic proteins like p62 directly interact with molecules involved in the TLR4-signaling pathway, but a correlation with the IRAK E3 ligase and scaffold protein Pellino3 remains obscure [3,4]. Hence, we are interested in elucidating the function of Pellino3 to prove our hypothesis that it is a key regulator in the TLR4-signaling cascade [5].

Methods

We used the cecal ligation and puncture (CLP) mouse model causing polymicrobial sepsis to analyze Pellino3 protein and mRNA expression. Furthermore, we induced endotoxemia in RAW264.7 mouse macrophages by LPS treatment to verify in vivo experiments. Lentiviral Pellino3 knockdown in RAW264.7 macrophages was used for cytokine measurements at mRNA level. To analyze potential Pellino3 binding partners in TLR4-signaling by mass spectrometry (MS), we overexpressed FLAG-tagged Pellino3 in RAW264.7 macrophages, treated cells for 3, 6 and 24 hours with LPS and immunoprecipitated Pellino3 via its FLAG-tag. To consider Pellino3 degradation as a result of p62-mediated autophagy, we transiently knocked down p62 by siRNA in RAW264.7 macrophages and also pharmacologically blocked LPS-induced autophagy by Bafilomycin A1.

Results

We demonstrated Pellino3 protein degradation in primary CD11b+ splenocytes after 24 hours following CLP operation and confirmed this in RAW264.7 macrophages after 24-hour LPS stimulation. Knockdown of Pellino3 attenuates proinflammatory cytokines, for example IL-6 mRNA, after 6 hours of LPS. Furthermore, we found by MS and verifying immunoprecipitation experiments that p62 is a Pellino3 binding partner, thus targeting Pellino3 for degradation. In line, both p62 knockdown and Bafilomycin A1 treatment prevent Pellino3 degradation, supporting an autophagic mechanism.

Conclusion

Our observations highlight a regulatory role of Pellino3 on TLR4 signaling. Thus, antagonism of Pellino3 in the hyperinflammatory phase of sepsis may counteract the cytokine storm. Furthermore, stabilization of Pellino3 by inhibition of autophagy in the hypoinflammatory phase of sepsis may improve immunity. In consideration of these two conflictive sepsis phases, modulation of Pellino3 may provide a new strategy for the development of a therapy approach in sepsis.

Acknowledgements

This research was supported by a grant from the Deutsche Forschungsgemeinschaft (KN493/9-1 and SFB TP3).

References

  1. Zhu J, Mohan C: Toll-like receptor signaling pathways - therapeutic opportunities.

    Mediators Inflamm 2010, 2010:781235. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  2. Xu Y, Liu XD, Gong X, Eissa NT: Signaling pathway of autophagy associated with innate immunity.

    Autophagy 2008, 4:110-112. PubMed Abstract | Publisher Full Text OpenURL

  3. Levine B, Mizushima N, Virgin HW: Autophagy in immunity and inflammation.

    Nature 2011, 469:323-335. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  4. Schauvliege R, Janssens S, Beyaert R: Pellino proteins are more than scaffold proteins in TLR/IL-1R signalling: a role as novel RING E3-ubiquitin-ligases.

    FEBS Lett 2006, 580:4697-4702. PubMed Abstract | Publisher Full Text OpenURL

  5. Hennessy EJ, Parker AE, O'Neill LA: Targeting Toll-like receptors: emerging therapeutics?

    Nat Rev Drug Discov 2010, 9:293-307. PubMed Abstract | Publisher Full Text OpenURL