DNA-PK is a DNA sensor for IRF-3-dependent innate immunity

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DNA-PK is a DNA sensor for IRF-3-dependent innate immunity

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DOI: http://dx.doi.org/10.7554/eLife.00047Published December 18, 2012 Cite as eLife 2012;1:e00047

Abstract

Innate immunity is the first immunological defence against pathogens. During virus infection detection of nucleic acids is crucial for the inflammatory response. Here we identify DNA-dependent protein kinase (DNA-PK) as a DNA sensor that activates innate immunity. We show that DNA-PK acts as a pattern recognition receptor, binding cytoplasmic DNA and triggering the transcription of type I interferon (IFN), cytokine and chemokine genes in a manner dependent on IFN regulatory factor 3 (IRF-3), TANK-binding kinase 1 (TBK1) and stimulator of interferon genes (STING). Both cells and mice lacking DNA-PKcs show attenuated cytokine responses to both DNA and DNA viruses but not to RNA or RNA virus infection. DNA-PK has well-established functions in the DNA repair and V(D)J recombination, hence loss of DNA-PK leads to severe combined immunodeficiency (SCID). However, we now define a novel anti-microbial function for DNA-PK, a finding with implications for host defence, vaccine development and autoimmunity.

DOI: http://dx.doi.org/10.7554/eLife.00047.001

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Acknowledgements

The authors thank Fred Alt for Prkdc+/− mice, Paul Hasty for supplying Xrcc5−/−/Trp53−/− MEFs and Xrcc5−/−/Xrcc6−/− murine skin fibroblasts, Penelope Jeggo and Brian Hemmings for Prkdc−/− MEFs, Shiuzu Akira for Dai−/− and Myd88/Trif−/− MEFs, Felix Randow for Tbk1−/− MEFs, Glen Barber for Sting−/− MEFs, Kate Fitzgerald for Irf3−/− and Mavs−/− MEFs, Wendy Barclay for NDV and influenza strain A/PR/8/34 and Colin Crump for HSV-1 strain S17. We thank Christian Ku for the production of the STING-293Trex cell line. Mass spectrometry was carried out by Dr Paul Hitchen at Imperial College Centre for Systems Biology. GLS is a Wellcome Trust Principal Research Fellow.

Decision letter

Ruslan Medzhitov, Reviewing editor, Yale University, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for choosing to send your work entitled "DNA-PK is a DNA sensor for IRF-3-dependent innate immunity" for consideration at eLife. Your article has been evaluated by a Senior Editor and 2 reviewers, 1 of whom is on the board of reviewing editors.

The Reviewing Editor and the other reviewer discussed their comments before we reached this decision, and the Reviewing Editor has assembled the following comments based on the reviewers' reports. While the reviewers found your work to be potentially interesting, there are some significant technical concerns. Specifically, more than one MEF line needs to be used to avoid MEF specific artifacts, and complementation experiments need to be performed to rule out contribution of cell transformation status and other possible effects specific to the MEF lines used. In addition, the mechanism of kinase activity independent effect of DNA-PK needs to be clarified – if it does function as a scaffolding protein, there should be some evidence provided to substantiate this hypothesis. The full comments follow:

1) Studies that are based on MEFs can sometimes be misleading due to MEF heterogeneity. Different preps of MEFs are not identical in their gene expression and behavior, and therefore it is critically important A) to test more than one preparation of MEFs to confirm all the key results and B) to perform complementation experiments by re-introducing the gene of interest, in this case DNA-PK, Ku70 and Ku80. It may be technically difficult to transfect DNA-PK because if its size, but Ku70 and Ku80 can be transfected to test recovery of the response to DNA and the virus. These experiments are particularly important because some of the innate immune responses to DNA are not present in transformed cells. Since transformed MEFs were used in this study, and the degree of transformation may be greater in the knockouts, it is important to perform careful complementation studies.

2) It is also surprising that the catalytic activity of DNA-PK is not required for the anti-viral response. The authors suggest that DNA-PK may function as a scaffold for signaling complex assembly. However, no evidence is provided here.

3) Finally, DNA-PK deficiency appears to affect some but not all DNA induced genes. Full gene expression analysis using microarray comparing WT and DNA-PK deficient cells would be very informative. In addition, comparison of gene expression in unstimulated WT and DNA-PK deficient MEFs (which would be part of a control for a microarray study) would help to address the issue of MEF heterogeneity and transformative state.

DOI: http://dx.doi.org/10.7554/eLife.00047.011

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