Mycobacterium abscessus Glycopeptidolipid Prevents Respiratory Epithelial TLR2 Signaling as Measured by HbD2 Gene Expression and IL-8 Release Lisa B. Davidson 1 , Rachid Nessar2, Prakasha Kempaiah3, Douglas J. Perkins3, Thomas F. Byrd1,4* 1 Department of Medicine, New Mexico Veterans Health Care System, Albuquerque, New Mexico, United States of America, 2 Universite Paris V-Descartes, Faculte´ de Medecine, Paris, France, 3 The University of New Mexico School of Medicine, Center for Global Health, Albuquerque, New Mexico, United States of America, 4 The University of New Mexico School of Medicine, Division of Infectious Diseases, Albuquerque, New Mexico, United States of America Abstract Mycobacterium abscessus has emerged as an important cause of lung infection, particularly in patients with bronchiectasis. Innate immune responses must be highly effective at preventing infection with M. abscessus because it is a ubiquitous environmental saprophyte and normal hosts are not commonly infected. M. abscessus exists as either a glycopeptidolipid (GPL) expressing variant (smooth phenotype) in which GPL masks underlying bioactive cell wall lipids, or as a variant lacking GPL which is immunostimulatory and invasive in macrophage infection models. Respiratory epithelium has been increasingly recognized as playing an important role in the innate immune response to pulmonary pathogens. Respiratory epithelial cells express toll-like receptors (TLRs) which mediate the innate immune response to pulmonary pathogens. Both interleukin-8 (IL-8) and human b-defensin 2 (HbD2) are expressed by respiratory epithelial cells in response to toll-like receptor 2 (TLR2) receptor stimulation. In this study, we demonstrate that respiratory epithelial cells respond to M. abscessus variants lacking GPL with expression of IL-8 and HbD2. Furthermore, we demonstrate that this interaction is mediated through TLR2. Conversely, M. abscessus expressing GPL does not stimulate expression of IL-8 or HbD2 by respiratory epithelial cells which is consistent with ‘‘masking’’ of underlying bioactive cell wall lipids by GPL. Because GPL-expressing smooth variants are the predominant phenotype existing in the environment, this provides an explanation whereby initial M. abscessus colonization of abnormal lung airways escapes detection by the innate immune system. Citation: Davidson LB, Nessar R, Kempaiah P, Perkins DJ, Byrd TF (2011) Mycobacterium abscessus Glycopeptidolipid Prevents Respiratory Epithelial TLR2 Signaling as Measured by HbD2 Gene Expression and IL-8 Release. PLoS ONE 6(12): e29148. doi:10.1371/journal.pone.0029148 Editor: Michael B. Fessler, National Institute of Environmental Health Sciences, United States of America Received July 24, 2011; Accepted November 21, 2011; Published December 21, 2011 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. Funding: TB receives support from the United States of America Department of Veterans Affairs, with this project funded by a University of New Mexico Research Allocation Committee Grant, and a grant from New Mexico Sonographics administered by the Biomedical Research Institute of New Mexico. 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: tbyrd@salud.unm.edu Introduction Mycobacterium abscessus, a nontuberculous mycobacterium, is an important emerging pathogen causing fibrocavitary lung disease which is often indistinguishable from disease caused by M. tuberculosis [1–3]. It is also an emerging infection in patients with cystic fibrosis [4–7]. Unlike M. tuberculosis, M. abscessus has the ability to express glycopeptidolipid (GPL) in the outer cell wall. Expression of GPL, or lack thereof, is correlated with the smooth or rough colony phenotype respectively, which is observed in certain species of nontuberculous mycobacteria [8]. GPL-mediat- ed biofilm formation is felt to facilitate survival in the environment and we have postulated that it may facilitate colonization of ectatic lung airways [8]. Spontaneous loss of GPL by M. abscessus is associated with acquisition of an invasive, immunostimulatory phenotype [8,9]. We have proposed that loss of GPL ‘‘unmasks’’ underlying bioactive cell wall lipids which mediate virulence [8,9]. Recently we deleted the gene mmpL4b, a gene coding for a critical enzyme in the biosynthetic GPL pathway [10,11], from the well characterized M. abscessus GPL-expressing variant 390S [8,9,12]. Assessing the interaction of this deletion mutant with human monocyte-derived macrophages, we demonstrated that loss of GPL is sufficient to convert this M. abscessus variant to a phenotype which is able to replicate in these cells and stimulate their toll-like receptors (TLRs) [13]. M. abscessus primarily causes lung disease in individuals who are immunosuppressed, or who have abnormal lung airways. Because M. abscessus is ubiquitous in the environment, innate immune responses must be highly effective in preventing infection as normal hosts are uncommonly infected. TLRs recognize patho- gen-associated molecular patterns and are the transducers of the innate immune response [14]. Mononuclear phagocytes have been extensively studied in terms of their TLR responses because they are actively involved in surveillance at the interface of the mucosal surfaces and the environment. Additionally, it has been recognized that respiratory epithelial cells lining the lung airways also play a critical role in surveillance and the innate immune response [15]. An important downstream effect of TLR signaling in respiratory epithelial cells is release of the chemokine interleukin-8 (IL-8) which recruits neutrophils from the circulation to the site(s) of TLR activation in the lung airways. As such, IL-8 release into cell supernates has been used as readout for TLR stimulation in experiments examining TLR responses of respiratory epithelial cells cultured in vitro. Human b-defensin (HbD2) is an antimicro- PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29148