TLR2/MyD88/NF-kB Pathway, Reactive Oxygen Species, Potassium Efflux Activates NLRP3/ASC Inflammasome during Respiratory Syncytial Virus Infection Jesus Segovia 1. , Ahmed Sabbah 1. , Victoria Mgbemena 1 , Su-Yu Tsai 1 , Te-Hung Chang 1 , Michael T. Berton 1 , Ian R. Morris 1 , Irving C. Allen 2 , Jenny P.-Y. Ting 2 , Santanu Bose 1 * 1 Department of Microbiology and Immunology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America, 2 Department of Microbiology-Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, United States of America Abstract Human respiratory syncytial virus (RSV) constitute highly pathogenic virus that cause severe respiratory diseases in newborn, children, elderly and immuno-compromised individuals. Airway inflammation is a critical regulator of disease outcome in RSV infected hosts. Although ‘‘controlled’’ inflammation is required for virus clearance, aberrant and exaggerated inflammation during RSV infection results in development of inflammatory diseases like pneumonia and bronchiolitis. Interleukin-1b (IL-1b) plays an important role in inflammation by orchestrating the pro-inflammatory response. IL-1b is synthesized as an immature pro-IL-1b form. It is cleaved by activated caspase-1 to yield mature IL-1b that is secreted extracellularly. Activation of caspase-1 is mediated by a multi-protein complex known as the inflammasome. Although RSV infection results in IL-1b release, the mechanism is unknown. Here in, we have characterized the mechanism of IL-1b secretion following RSV infection. Our study revealed that NLRP3/ASC inflammasome activation is crucial for IL-1b production during RSV infection. Further studies illustrated that prior to inflammasome formation; the ‘‘first signal’’ constitutes activation of toll-like receptor-2 (TLR2)/MyD88/NF-kB pathway. TLR2/MyD88/NF-kB signaling is required for pro- IL-1b and NLRP3 gene expression during RSV infection. Following expression of these genes, two ‘‘second signals’’ are essential for triggering inflammasome activation. Intracellular reactive oxygen species (ROS) and potassium (K + ) efflux due to stimulation of ATP-sensitive ion channel promote inflammasome activation following RSV infection. Thus, our studies have underscored the requirement of TLR2/MyD88/NF-kB pathway (first signal) and ROS/potassium efflux (second signal) for NLRP3/ASC inflammasome formation, leading to caspase-1 activation and subsequent IL-1b release during RSV infection. Citation: Segovia J, Sabbah A, Mgbemena V, Tsai S-Y, Chang T-H, et al. (2012) TLR2/MyD88/NF-kB Pathway, Reactive Oxygen Species, Potassium Efflux Activates NLRP3/ASC Inflammasome during Respiratory Syncytial Virus Infection. PLoS ONE 7(1): e29695. doi:10.1371/journal.pone.0029695 Editor: David M. Ojcius, University of California Merced, United States of America Received September 14, 2011; Accepted December 2, 2011; Published January 25, 2012 Copyright: ß 2012 Segovia 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 work was supported by the National Institutes of Health (NIH) grants AI083387 (SB), AI057986 (MTB), U19 AI077437 (JPYT), U54 AI057157 (JPYT) and a grant from Center for Innovation in Prevention and Treatment of Airway Diseases (CIPTAD) (SB). AS and VM were supported by NIH National Institute of Dental and Craniofacial Research (NIDCR) grant DE14318 for the COSTAR program. IRM was supported by NIH training grant T32AI007271. The funders 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: bose@uthscsa.edu . These authors contributed equally to this work. Introduction Human respiratory syncytial virus (RSV) is a RNA respiratory virus that infects lung epithelial cells to cause high mortality and morbidity among infants, children and elderly by developing severe respiratory diseases like pneumonia and bronchiolitis [1–3]. These diseases occur due to massive and ‘‘uncontrolled’’ inflammation of the respiratory tract. It is believed that prolonged virus infection (and resulting high viral replication/multiplication) results in a robust inflammatory response that is detrimental to the infected host. The innate immune antiviral response is the first line of defense against virus infection before induction of the adaptive immune response [4– 6]. It is well established that innate response is critical to restrict virus spread and infection resulting in diminished disease burden. The inflammatory response constitutes a critical innate defense mecha- nism triggered by the host to control infections [7]. However, aberrant and unregulated inflammation results in development of various disease states including pneumonia and bronchiolitis. Interleukin-1b (IL-1b) is a critical cytokine that acts as a pyrogen to amplify the pro-inflammatory response during infection with various pathogens. IL-1b produced from infected cells acts via an autocrine/paracrine mechanism to activate NF- kB/MAP kinase dependent pro-inflammatory cytokines and chemokines to establish an effective immune response for combating infection. Respiratory RNA viruses like RSV and influenza A virus induce secretion of IL-1b in the respiratory tract during infection of mouse and humans and its secretion is critical for ‘‘shaping’’ the anti-viral inflammatory response to clear virus from the airway [8–12]. Production of IL-1b from macrophages requires three steps – a) expression of pro-IL-1b gene and synthesis of immature pro-IL-1b protein, b) processing (cleavage) of pro-IL- 1b by active caspase-1 to yield the mature form of IL-1b, and c) secretion of mature IL-1b from the cell to the extracellular environment via Rab-3a containing secretory vesicle [13]. Generation of mature IL-1b is achieved following cytoplasmic assembly and activation of inflammasomes [14–19]. The NLR PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e29695