Endothelial IL-33 Expression Is Augmented by Adenoviral Activation of the DNA Damage Machinery This information is current as Tor Espen Stav-Noraas, Reidunn J. Edelmann, Lars La Cour of September 29, 2021. Poulsen, Olav Sundnes, Danh Phung, Axel M. Küchler, Fredrik Müller, Amine A. Kamen, Guttorm Haraldsen, Mari Kaarbø and Johanna Hol J Immunol 2017; 198:3318-3325; Prepublished online 3 March 2017; Downloaded from doi: 10.4049/jimmunol.1600054 http://www.jimmunol.org/content/198/8/3318 Supplementary http://www.jimmunol.org/content/suppl/2017/03/03/jimmunol.160005 http://www.jimmunol.org/ Material 4.DCSupplemental References This article cites 52 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/198/8/3318.full#ref-list-1 Why The JI? Submit online. by guest on September 29, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Endothelial IL-33 Expression Is Augmented by Adenoviral Activation of the DNA Damage Machinery Tor Espen Stav-Noraas,*,† Reidunn J. Edelmann,*,† Lars La Cour Poulsen,*,† Olav Sundnes,*,† Danh Phung,† Axel M. Kuchler,€ † Fredrik Muller,€ ‡ Amine A. Kamen,x Guttorm Haraldsen,*,† Mari Kaarbø,‡,1 and Johanna Hol*,†,1 IL-33, required for viral clearance by cytotoxic T cells, is generally expressed in vascular endothelial cells in healthy human tissues. We discovered that endothelial IL-33 expression was stimulated as a response to adenoviral transduction. This response was de- pendent on MRE11, a sensor of DNA damage that can also be activated by adenoviral DNA, and on IRF1, a transcriptional regulator of cellular responses to viral invasion and DNA damage. Accordingly, we observed that endothelial cells responded to adenoviral DNA by phosphorylation of ATM and CHK2 and that depletion or inhibition of MRE11, but not depletion of ATM, abrogated IL-33 stimulation. In conclusion, we show that adenoviral transduction stimulates IL-33 expression in endothelial cells in a manner that Downloaded from is dependent on the DNA-binding protein MRE11 and the antiviral factor IRF1 but not on downstream DNA damage response signaling. The Journal of Immunology, 2017, 198: 3318–3325. member of the IL-1 family, IL-33 (1, 2) appears to be synthetic analog of viral dsRNA) by TLR3 in murine hepatocytes crucially involved in establishing a successful antiviral (7) and human fibroblasts (8). In addition, synthesis of IL-33 is A CD8 T cell response in the mouse (3). Viral infection strongly boosted in human fibroblasts when polyinosinic- http://www.jimmunol.org/ also drives expression of IL-33 in many contexts. For example, polycytidylic acid acts in concert with TGF-b (8). murine lungs infected with influenza A show a dramatic increase Host recognition of viral infection involves several classes of in IL-33 expression (4), and patients with chronic viral hepatitis sensors, including TLRs, C-type lectins, cytosolic RNA or DNA have elevated serum levels of IL-33 (5). These observations trig- sensors, and the nuclear MRN complex (consisting of MRE11, gered interest in understanding how IL-33 expression is regulated NBS1, and RAD50) (9–11). This complex is well characterized at the cellular level. For example, transcription of IL-33 in murine as an initiator of the DNA damage response. The DNA damage macrophages partially depends on activation of IRF3 via the RNA response is crucial to prevent replication of damaged genomic sensor retinoic acid inducible gene I (6). IL-33 synthesis can also host material, but it also serves to recognize foreign DNA. by guest on September 29, 2021 be triggered by detection of polyinosinic-polycytidylic acid (a Human adenovirus 5 (Ad5) has a 36-kb dsDNA genome that is replicated concomitantly with cellular DNA. Thus, the dis- *K.G. Jebsen Inflammation Research Centre, Oslo University Hospital and University of covery that Ad5 early proteins interfere with DNA damage † Oslo, N-0424 Oslo, Norway; Laboratory of Immunohistochemistry and Immunopathology, response mediators elicited great interest, suggesting that the Department of Pathology, Oslo University Hospital and University of Oslo, N-0424 Oslo, Norway; ‡Department of Microbiology, Oslo University Hospital and University of Oslo, cellular DNA damage response also plays an antiviral role x N-0424 Oslo, Norway; and Department of Bioengineering, McGill University, Montreal, (discussed in Ref. 11). Indeed, adenovirus targets the MRN Quebec H3A OC3, Canada complex for proteasomal degradation by expressing the early 1 M.K. and J.H. contributed equally to this work. proteins E1b55k/E4orf6 and E4orf3, thus limiting activation of ORCIDs: 0000-0003-1554-4775 (T.E.S.-N.); 0000-0001-9077-164X (R.J.E.); 0000- the DNA damage machinery in response to adenoviral DNA 0002-7266-3108 (L.L.C.P.); 0000-0002-4930-3227 (O.S.); 0000-0001-8725- 1563 (D.P.); 0000-0003-4512-1079 (A.M.K.); 0000-0003-0812-0640 (F.M.); 0000- (12). In the absence of adenoviral E4 proteins, MRN associates 0001-9110-8815 (A.A.K.); 0000-0001-5837-5006 (J.H.). with viral DNA and initiates repair processes that result in Received for publication January 12, 2016. Accepted for publication February 6, tethering of viral linear DNA (concatemer formation) and 2017. prevent viral replication (12, 13). This work was supported by grants from Helse Sør-Øst (2010051, 2010019, 2013115, 2014032), the Research Council of Norway (221929/F20), and the University of Oslo Although the in vivo importance of IL-33 in antiviral defense (131406). was highlighted experimentally in mice (3), significant differences T.E.S.-N. and R.J.E. designed and performed experiments, interpreted data, and wrote in the distribution of IL-33 between mouse and human may point the manuscript; L.L.C.P., O.S., A.M.K., and D.P. designed and performed experiments, to species-specific functions. For example, although IL-33 is interpreted data, and critically revised the manuscript; A.A.K. and F.M. provided virus and critically revised the manuscript; and G.H., M.K., and J.H. designed experiments, nearly absent from vascular endothelial cells in the mouse, most interpreted data, and wrote and critically revised the manuscript. All authors approved IL-33 in healthy human tissues is found in the vasculature (14– the final version of the submitted manuscript. 16). It is unclear whether this vascular pool of IL-33 has a function Address correspondence and reprint requests to Prof. Guttorm Haraldsen, Oslo Uni- in antiviral immune defense that cannot be accounted for in mu- versity Hospital and University of Oslo, Post Box 4950 Nydalen, 0424 Oslo, Norway. E-mail address: [email protected] rine models. The online version of this article contains supplemental material. We report that nonreplicative Ad5 (nrAd5) increases endothelial expression of IL-33 and initiates a DNA damage response. De- Abbreviations used in this article: Ad5, adenovirus 5; csNICD1, cleaved NOTCH1 intracellular domain; DAPT, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine pletion of MRE11 or IRF1 [essential transcriptional regulator of the t-butyl ester; hpt, h posttransduction; moi, multiplicity of infection; nrAd5, nonreplicative DNA damage response (17)] prevented the observed stimulation of Ad5; qRT-PCR, quantitative RT-PCR; siRNA, small interfering RNA. IL-33, implying that sensing of viral DNA by MRE11 boosts Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 endothelial IL-33 expression. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600054 The Journal of Immunology 3319 Materials and Methods IFN-a/b neutralization Cell culture and reagents In vitro blocking of the IFN-a/b receptor was performed with a murine mAb to Umbilical cords were obtained from the Department of Gynecology and human IFN-a/b receptor chain 2 (MMHAR-2, 10 mg/ml). A species-, isotype-, Obstetrics at the Oslo University Hospital, according to a protocol approved and concentration-matched mAb against the E-tag epitope (Supplemental by the Regional Committee for Research Ethics (S-05152a). HUVECs were Table I, 10 mg/ml) was used as a negative control. Ad5DE1DE3-GFP (50 isolated as described by Jaffe et al. (18) and cultured in MCDB 131 medium moi) was added to the culture, and the cells were incubated for 24 h. The results were compared with those from nontreated and nontransduced cells. To (Life Technologies) containing 7.5% FCS, 5 mM L-glutamine (Invitrogen), 10 ng/ml epidermal growth factor, 1 ng/ml basic fibroblast growth factor test the efficacy of the neutralizing Ab, CXCL10 was measured in isotype- and (both from R&D Systems), 1 mg/ml hydrocortisone (Sigma-Aldrich), MMHAR-2–treated cells stimulated with 1000 U/ml IFN-a. 50 mg/ml gentamycin, and 250 ng/ml amphotericin B (both from Quantitative RT-PCR Lonza), unless otherwise stated. Cells were used at passages one to six, maintained at 37˚C in 95% humidity/5% CO2 atmosphere, and split at a 1:3 Upon harvest, total RNA was extracted from cells using TRIzol Reagent ratio. The g-secretase inhibitor N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S- (Thermo Fisher Scientific), according to the manufacturer’s recommen- phenylglycine t-butyl ester (DAPT; EMD Chemicals) was dissolved in dations.