NADPH Oxidase 1 Is Associated with Altered Host Survival and T Cell Phenotypes after Influenza A Virus Infection in Mice

RESEARCH ARTICLE NADPH Oxidase 1 Is Associated with Altered Host Survival and T Cell Phenotypes after Influenza A Virus Infection in Mice Amelia R. Hofstetter1,2, Juan A. De La Cruz1, Weiping Cao1, Jenish Patel1, Jessica A. Belser1, James McCoy2, Justine S. Liepkalns1, Samuel Amoah1, Guangjie Cheng2¤, Priya Ranjan1, Becky A. Diebold2, Wun-Ju Shieh3, Sherif Zaki3, Jacqueline M. Katz1, Suryaprakash Sambhara1, J. David Lambeth2*, Shivaprakash Gangappa1* 1 Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 2 Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia, United States of America, 3 Infectious Disease Pathology Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America ¤ Current address: Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, UAB School of Medicine, Birmingham, Alabama, United States of America * (SG); (JDL) OPEN ACCESS Citation: Hofstetter AR, De La Cruz JA, Cao W, Patel J, Belser JA, McCoy J, et al. (2016) NADPH Oxidase 1 Is Associated with Altered Host Survival and T Cell Phenotypes after Influenza A Virus Infection in Mice. PLoS ONE 11(2): e0149864. doi:10.1371/journal. pone.0149864 Editor: Bumsuk Hahm, University of MissouriColumbia, UNITED STATES Received: October 14, 2015 Accepted: February 5, 2016 Published: February 24, 2016 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study was funded by National Institutes of Health Grants AI102197-01 (JDL) (http:// report.nih.gov/quicklinks.aspx), T32 DK00771 (http:// report.nih.gov/quicklinks.aspx), and Centers for Disease Control and Prevention intramural funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract The role of the reactive oxygen species-producing NADPH oxidase family of enzymes in the pathology of influenza A virus infection remains enigmatic. Previous reports implicated NADPH oxidase 2 in influenza A virus-induced inflammation. In contrast, NADPH oxidase 1 (Nox1) was reported to decrease inflammation in mice within 7 days post-influenza A virus infection. However, the effect of NADPH oxidase 1 on lethality and adaptive immunity after influenza A virus challenge has not been explored. Here we report improved survival and decreased morbidity in mice with catalytically inactive NADPH oxidase 1 (Nox1*/Y) compared with controls after challenge with A/PR/8/34 influenza A virus. While changes in lung inflammation were not obvious between Nox1*/Y and control mice, we observed alterations in the T cell response to influenza A virus by day 15 post-infection, including increased interleukin-7 receptor-expressing virus-specific CD8+ T cells in lungs and draining lymph nodes of Nox1*/Y, and increased cytokine-producing T cells in lungs and spleen. Furthermore, a greater percentage of conventional and interstitial dendritic cells from Nox1*/Y draining lymph nodes expressed the co-stimulatory ligand CD40 within 6 days post-infection. Results indicate that NADPH oxidase 1 modulates the innate and adaptive cellular immune response to influenza virus infection, while also playing a role in host survival. Results suggest that NADPH oxidase 1 inhibitors may be beneficial as adjunct therapeutics during acute influenza infection. PLOS ONE | DOI:10.1371/journal.pone.0149864 February 24, 2016 1 / 19 Role of Nox1 Oxidase after Influenza A Virus Infection Competing Interests: The authors have declared that no competing interests exist. Introduction Despite extensive influenza virus surveillance and seasonal influenza vaccination coverage, influenza A virus (IAV) remains a major threat to public health. Seasonal influenza viruses cause illness in 2–5 million individuals annually, and 250,000–500,000 will succumb to complications from the disease [1]. Furthermore, the continual reassortment of IAVs within wild birds and domestic animals drives the occasional emergence of avian or swine influenza viruses that can infect humans [2, 3]. Some of these prove to be highly pathogenic, such as H5N1 and H7N9, which are fatal in 20–60% of individuals [4]. In the majority of lethal cases of influenza, death is attributed to acute respiratory distress syndrome [5], a more severe form of acute lung injury [6]. Current efforts to combat death related to IAV infection target the virus: vaccination and antiviral therapy. Both of these approaches are vulnerable to loss of efficacy due to viral mutations [7, 8]. Furthermore, several lines of investigation have implicated the host immune system as a contributing factor to pathology [9–11]. Along with vaccination and antivirals, there has been interest in development of adjunct therapeutics to decrease the inflammatory processes that underlie acute lung injury/acute respiratory distress syndrome by targeting the host immune system [12–14]. Such a strategy has been shown to improve outcomes of IAV infection in mouse models [10, 15, 16] and in the clinic [17]. These results underscore the potential of adjunct therapeutics to decrease the disease burden of IAV. Reactive oxygen species (ROS) have been implicated in the lung pathology associated with severe cases of seasonal or pandemic IAV [18–24]. Superoxide produced by NADPH oxidase 2 (Nox2) has been shown to contribute to influenza-mediated lung pathology [23, 25, 26]. However, other sources of ROS in the lung include the Nox1 and Nox4 isoenzymes, as well as the closely related dual oxidase enzymes (Duox1 and Duox2), all of which are expressed by alveolar epithelial cells [27, 28]. In a previous study, Nox1 was shown to modulate influenza-induced inflammation in the early phase (days 3–7) post-infection (p.i.) with a non-lethal dose of influenza [29]. However, the influence of Nox1 after a lethal challenge of influenza infection has not been reported. It is also unclear how Nox1 might contribute to the development of adaptive immune responses following influenza virus clearance. In this study, we demonstrate that mice expressing an inactive form of Nox1 (Nox1 /Y mice) [30] have improved survival after IAV challenge compared with C57BL/6 control mice. We also observed alterations to the adaptive immune response after IAV challenge, including a decreased percentage of virus-specific CD8+ T cells in the lungs, an increased percentage of virus-specific CD8+ T cells expressing the IL-7 receptor (CD127) in the lungs and draining lymph nodes, and an increased percentage of T cells in the lung and spleen with cytokine effector function ex vivo in Nox (...truncated)