Original Contribution Antibodies against dengue virus nonstructural protein-1 induce heme oxygenase-1 via a redox-dependent pathway in human endothelial cells Stephan Immenschuh a,n,1 , Puji Rahayu b,1 , Behnat Bayat b , Hendry Saragih a,c , Andhika Rachman d , Sentot Santoso b,n a Institute for Transfusion Medicine, Hannover Medical School, 30625 Hannover, Germany b Institute for Clinical Immunology and Transfusion Medicine, Justus-Liebig University, 35385 Giessen, Germany c Faculty of Biology, Gadjah Mada University, Yogyakarta, Indonesia d Department of Oncology and Hematology, University of Indonesia, Jakarta, Indonesia article info Article history: Received 6 July 2012 Received in revised form 11 October 2012 Accepted 18 October 2012 Available online 26 October 2012 Keywords: Heme oxygenase-1 Endothelium Redox signaling Dengue fever Anti-endothelial antibodies Free radicals abstract Heme oxygenase (HO)-1, the inducible isoform of the first and rate-limiting enzyme of heme degradation, affords anti-inflammatory protection via its cell-type-specific effects in endothelial cells (ECs). In dengue hemorrhagic fever (DHF), which is the life-threatening form of dengue virus (DV) infection, endothelial interactions of cross-reactive antibodies against the DV nonstructural glycoprotein-1 (NS1) are associated with endothelial dysfunction. In this study, we investigated whether anti-NS1 antibodies might regulate HO-1 gene expression in human ECs. Serum from DHF patients with high anti- NS1 titers and a monoclonal anti-NS1 antibody upregulated HO-1 gene expression in human umbilical vein ECs, which was blocked by purified NS1 antigen. Immunoprecipitation studies showed that anti-NS1 antibodies specifically bound to the oxidoreductase protein disulfide isomerase (PDI) on ECs. Moreover, anti-NS1-mediated HO-1 induction was reduced by inhibition of PDI enzyme activity. Reactive oxygen species, which were generated by NADPH oxidase and in turn activated the phosphatidylinositol 3-kinase (PI3K)/Akt cascade, were involved in this upregulation of HO-1 gene expression. Finally, apoptosis of ECs caused by anti-NS1 antibodies was increased by pharmacological inhibition of HO-1 enzyme activity. In conclusion, HO-1 gene expression is upregulated by anti-NS1 antibodies via activation of a redox- dependent PDI/PI3K/Akt-mediated pathway in human ECs. & 2012 Elsevier Inc. All rights reserved. Heme oxygenase (HO)-1 2 is the inducible isoform of HO and catalyzes the enzymatic degradation of free heme to produce iron, carbon monoxide (CO), and biliverdin, which is converted into bilirubin via a coupled reaction [1,2]. Studies in HO-1 knockout mice and in a human individual with genetic HO-1 deficiency have revealed that HO-1 not only protects against oxidative stress, but also plays a crucial role in the pathogenesis of inflammation [3–8]. In particular, HO-1 has been demonstrated to afford protection in various cardiovascular disorders by its anti-inflammatory endothelial cell (EC)-specific effects [9–11]. Gene expression of HO-1 is upregulated by a host of stress- dependent and -independent stimuli in various types of cells and tissues in vivo and in vitro [12,13]. Much less, however, is known about the specific regulation of HO-1 gene expression in the endothelium. More recently, HO-1 has been shown to be induced by ligation of anti-endothelial cell antibodies (AECAs) that are directed against human leukocyte antigens (HLAs) in ECs [14,15]. To further investigate whether AECAs may play a role in EC- specific regulation of HO-1, we examined the regulatory effects of antibodies against the dengue virus nonstructural protein-1 (NS1) on HO-1 gene expression in human umbilical vein endothelial cells (HUVECs). Dengue is a major arthropod-borne viral disease in tropical countries and is transmitted by the Aedes aegyptii and Aedes albopictus mosquitoes. Dengue is caused by infection with dengue virus (DV) [16] and can lead to the life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome, both of which are characterized by vascular hemorrhage and capillary Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine 0891-5849/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2012.10.551 n Corresponding authors. Fax: þ49 511 532 2079 (S. Immenschuh) or þ49 64198541529 (S. Santoso). E-mail addresses: Immenschuh.Stephan@mh-hannover.de (S. Immenschuh), sentot.santoso@immunologie.med.uni-giessen.de (S. Santoso). 1 These authors contributed equally to this work. 2 Abbreviations used: AECA, anti-endothelial cell antibody; CoPP, Co-protoporphyrin IX; DF, dengue fever; H 2 DCFDA, 5,6-carboxy-2 0 ,7 0 -dichlorodi- hydrofluorescein diacetate; DHF, dengue hemorrhagic fever; DPI, diphenyliodo- nium; DV, dengue virus; EC, endothelial cell; FCS, fetal calf serum; GAPDH, glyceraldehyde dehydrogenase; HO, heme oxygenase; HUVEC, human umbilical vein endothelial cell; mab, monoclonal antibody; NAC, N-acetylcysteine; NS1, nonstructural protein-1; PDI, protein disulfide isomerase; PI3K, phosphatidylino- sitol 3-kinase; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; SnMP, Sn-mesoporphyrin. Free Radical Biology and Medicine 54 (2013) 85–92