The Nitric Oxide Pathway Provides Innate Antiviral Protection in Conjunction with the Type I Interferon Pathway in Fibroblasts Devangi R. Mehta, Ali A. Ashkar, Karen L. Mossman* Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre, Michael DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada Abstract The innate host response to virus infection is largely dominated by the production of type I interferon and interferon stimulated genes. In particular, fibroblasts respond robustly to viral infection and to recognition of viral signatures such as dsRNA with the rapid production of type I interferon; subsequently, fibroblasts are a key cell type in antiviral protection. We recently found, however, that primary fibroblasts deficient for the production of interferon, interferon stimulated genes, and other cytokines and chemokines mount a robust antiviral response against both DNA and RNA viruses following stimulation with dsRNA. Nitric oxide is a chemical compound with pleiotropic functions; its production by phagocytes in response to interferon-c is associated with antimicrobial activity. Here we show that in response to dsRNA, nitric oxide is rapidly produced in primary fibroblasts. In the presence of an intact interferon system, nitric oxide plays a minor but significant role in antiviral protection. However, in the absence of an interferon system, nitric oxide is critical for the protection against DNA viruses. In primary fibroblasts, NF-kB and interferon regulatory factor 1 participate in the induction of inducible nitric oxide synthase expression, which subsequently produces nitric oxide. As large DNA viruses encode multiple and diverse immune modulators to disable the interferon system, it appears that the nitric oxide pathway serves as a secondary strategy to protect the host against viral infection in key cell types, such as fibroblasts, that largely rely on the type I interferon system for antiviral protection. Citation: Mehta DR, Ashkar AA, Mossman KL (2012) The Nitric Oxide Pathway Provides Innate Antiviral Protection in Conjunction with the Type I Interferon Pathway in Fibroblasts. PLoS ONE 7(2): e31688. doi:10.1371/journal.pone.0031688 Editor: William P. Halford, Southern Illinois University School of Medicine, United States of America Received January 4, 2012; Accepted January 11, 2012; Published February 21, 2012 Copyright: 2012 Mehta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by a Canadian Institutes of Health Research operating grant (MOP 57669) to KLM. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mossk@mcmaster.ca Introduction A central theme today in medical research is understanding the delicate balance of interactions between a pathogen and its host. These interactions dictate the pathological consequences of infections. It is well recognized that the innate immune response against pathogens focuses on detection of highly conserved pathogen-associated molecular patterns (PAMPs) that are distinct from the host. Host pathogen recognition receptors have evolved to recognize patterns such as nucleic acids from pathogens including bacteria and viruses [1]. The first line of defence against invading pathogens is the rapid and robust production of type I interferon (IFN), a family of cytokines with potent immune stimulatory and pathogen-controlling properties. Fibroblasts are amongst the first cell types involved in the line of defence against numerous pathogens. Fibroblasts are widely distributed in organisms [2] and play an important role in the transition from innate to adaptive immunity [3,4]. This role is largely a result of cytokine production [5], most notably IFNb, which was originally termed fibroblast IFN. As such, fibroblasts are important effectors of the early innate immune response. Indeed, in a recent study, non-hematopoietic cells, and fibroblasts in particular, were shown to mediate protection against an emerging viral infection through a type I IFN response [6]. As all viruses are thought to make double-stranded RNA (dsRNA) as a by-product of their replication cycles, this molecule is a potent producer of type I IFN and as such, is commonly used to study innate immune responses to virus infection. DsRNA can be recognized by three different families of pathogen recognition receptors, the toll-like receptors (TLRs), the retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) and the nucleotide oligomer- ization domain-like receptors (NLRs); these bind dsRNA and initiate cellular signaling pathways [7,8]. The TLRs and RLRs elicit antiviral pathways involving type I and type III IFNs and cytokine production, whereas NLRs elicit caspase 1 activation for IL-1b maturation [7,8]. In fibroblasts, IFN is typically made upon detection of viral dsRNA by TLR-3 and the RLRs, RIG-I and melanoma differentiation-associated gene 5 (MDA5). These pathways converge on NF-kB and IFN regulatory factor 3 (IRF3), which, upon activation, are important for cytokine and IFNb production. Type I IFN, such as IFNb, signal through the JAK-STAT pathway, which includes IRF9 as an essential component. This signaling leads to the induction of IRF7, which amplifies the cellular antiviral response through the generation of IFNa species and IFN stimulated genes (ISGs) [8]. In the prototypic antiviral response, IRF3 and early production of type I IFN and ISGs are arguably the most important events in combating infection. In humans, inborn errors that impair the PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e31688