Correspondence

by the 2011 Nobel Prize in Physiology or Medicine (4). How should the scientific community resolve this paradox? The cross-species comparison of expression arrays by Seok and colleagues has injected new, important data into this ongoing conversation (2). Proving the absence of an association between mouse and human is difficult, as highlighted by the methodological issues raised in Perlman and colleagues’ editorial (1). Given the profound implications of Seok and colleagues’ article, clinicians and researchers interested in critical illness would have benefited from an even more intense effort by the authors to compare “apples to apples.” We propose that two additional experiments would have strengthened their conclusions, particularly regarding the dissimilarity in leukocyte responses to endotoxin. First, Seok and colleagues showed that the mouse transcriptional response to endotoxin was markedly attenuated immediately downstream of TLR4 (2). Because the murine version of TLR4 is considered the true ortholog of the human gene, this surprising result made us wonder whether an adequate exposure to endotoxin was achieved or whether the transcriptional signature of high-TLR4expressing cells in mice (e.g., monocytes) was swamped out by lower-expressing cells (e.g., lymphocytes). Had Seok and colleagues been able to adjust endotoxin dose or duration to achieve comparable TLR4 signaling responses in their own readout, the subsequent negative findings would have been greatly bolstered. Second, Seok and colleagues could have compared transcriptional responses to endotoxin of homologous cell types between species—for example, monocytes isolated from each species stimulated with the same endotoxin serotype analyzed at comparable doses and durations of exposure. Had the ex vivo profiles been unexpectedly similar, readers could then have interpreted the in vivo dissimilarity more deeply, for example, by raising the possibility that mouse and human responses to endotoxin do not diverge at the initial molecular events, but rather, in their subsequent effects on the intercellular network found within the body. Overall, we commend Seok and colleagues on their nuanced approach to the mouse-as-sepsis-model question. We believe that ample evidence exists to support use of the mouse as a discovery tool, but that future mechanistic studies in sepsis should emphasize early validation in the human setting (5). Rather than delaying proof-of-concept human studies, basic and clinical researchers in this field should collaborate early and extensively to ensure that the most “translatable” science is moving forward. Author disclosures are available with the text of this letter at www.atsjournals.org.

S. Ananth Karumanchi, M.D. Howard Hughes Medical Institute Chevy Chase, Maryland and Beth Israel Deaconess Medical Center and Harvard Medical School Boston, Massachusetts Samir M. Parikh, M.D. Beth Israel Deaconess Medical Center and Harvard Medical School Boston, Massachusetts References 1. Perlman H, Budinger GRS, Ward PA. Humanizing the mouse: in defense of murine models of critical illness [editorial]. Am J Respir Crit Care Med 2013;187:898–900. 2. Seok J, Warren HS, Cuenca AG, Mindrinos MN, Baker HV, Xu W, Richards DR, McDonald-Smith GP, Gao H, Hennessy L, et al.; Inflammation and Host Response to Injury, Large Scale Collaborative Research Program. Genomic responses in mouse models poorly

1265 mimic human inflammatory diseases. Proc Natl Acad Sci USA 2013; 110:3507–3512. 3. Opal SM, Laterre PF, Francois B, LaRosa SP, Angus DC, Mira JP, Wittebole X, Dugernier T, Perrotin D, Tidswell M, et al.; ACCESS Study Group. Effect of eritoran, an antagonist of MD2-TLR4, on mortality in patients with severe sepsis: the ACCESS randomized trial. JAMA 2013;309:1154–1162. 4. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, et al. Defective LPS signaling in C3H/ HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998;282: 2085–2088. 5. David S, Mukherjee A, Ghosh CC, Yano M, Khankin EV, Wenger JB, Karumanchi SA, Shapiro NI, Parikh SM. Angiopoietin-2 may contribute to multiple organ dysfunction and death in sepsis. Crit Care Med 2012;40: 3034–3041. Copyright ª 2013 by the American Thoracic Society

Reply From the Editorialists: Drs. Karumanchi and Parikh highlight the conflicting effects of activation of the Toll-like receptor 4 by LPS on peripheral blood leukocyte gene expression in mice and patients. This analysis is limited by many of the same methodological concerns raised in our editorial (1). In addition, because of safety concerns, the dose of LPS that Seok and colleagues (2) used to treat mice was w100,000-fold less than the lethal dose in mice (w40 mg/kg), and a correspondingly low dose was administered to humans. Thus, the relevance of these studies to patients with sepsis is limited. The issue of the contribution of Toll-like receptor signaling in sepsis will be difficult to unravel as these receptors are likely to be activated by both primary (pathogen-related) and secondary (neutrophil extracellular traps and release of mitochondrial DNA) events that contribute to the pathogenesis of sepsis (3). The suggestion by Karumanchi and Parikh to use cultured human cells is one valid approach; however, culturing immune cells on plastic and other artificial factors imposed by ex vivo culture may skew the immune response of these cells. Therefore, we continue to stress the need for development of novel animal model systems, including the humanized mouse and others as preclinical models of human sepsis (4). Author disclosures are available with the text of this letter at www.atsjournals.org.

Harris Perlman, Ph.D. G. R. Scott Budinger, M.D. Northwestern University Chicago, Illinois Peter A. Ward, M.D. University of Michigan Ann Arbor, Michigan

References 1. Perlman H, Budinger GRS, Ward PA. Humanizing the mouse: in defense of murine models of critical illness [editorial]. Am J Respir Crit Care Med 2013;187:898–900. 2. Seok J, Warren HS, Cuenca AG, Mindrinos MN, Baker HV, Xu W, Richards DR, McDonald-Smith GP, Gao H, Hennessy L, et al.; Inflammation and Host Response to Injury, Large Scale Collaborative Research Program. Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci USA 2013;110:3507– 3512.

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3. Kawai T, Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 2011;34:637–650. 4. Shultz LD, Brehm MA, Garcia-Martinez JV, Greiner DL. Humanized mice for immune system investigation: progress, promise and challenges. Nat Rev Immunol 2012;12:786–798. Copyright ª 2013 by the American Thoracic Society

Obesity Paradox? To the Editor :

The obesity paradox is a phrase often used to describe the curious epidemiological finding that obese individuals are somehow predisposed to the development of disease, but outcomes once the disease is established are improved when compared with lean counterparts. The obesity paradox has been described in association with various cardiovascular conditions, but recently, a similar phenomenon was reported in patients with acute lung injury (1–3). However, because of the complexity of reading chest radiographs from obese individuals (e.g., because of atelectasis, increased soft-tissue density, and low lung volumes) many experts believe that “predisposition” as well as “improved outcomes” may be explained, at least in part, by the fact that acute lung injury is overdiagnosed in obese individuals. It is for this reason I was disappointed to find out that body mass index was not included in the excellent study by Thille and colleagues published in the Journal (4). If the authors collected data on height and weight, could they kindly evaluate whether an inverse association between obesity and diffuse alveolar damage pathology was observed in their study? Author disclosures are available with the text of this letter at www.atsjournals.org.

Ross S. Summer, M.D. Thomas Jefferson University Philadelphia, Pennsylvania

References 1. Gong MN, Bajwa EK, Thompson BT, Christiani DC. Body mass index is associated with the development of acute respiratory distress syndrome. Thorax 2010;65:44–50. 2. Morris AE, Stapleton RD, Rubenfeld GD, Hudson LD, Caldwell E, Steinberg KP. The association between body mass index and clinical outcomes in acute lung injury. Chest 2007;131:342–348. 3. O’Brien JM Jr, Welsh CH, Fish RH, Ancukiewicz M, Kramer AM; National Heart, Lung, and Blood Institute Acute Respiratory Distress Syndrome Network. Excess body weight is not independently associated with outcome in mechanically ventilated patients with acute lung injury. Ann Intern Med 2004;140:338–345. 4. Thille AW, Esteban A, Fernández-Segoviano P, Rodriguez J, Aramburu J, Pen˜uelas O, Cortés-Puch I, Cardinal-Fernández P, Lorente JA, FrutosVivar F. Comparison of the Berlin definition for acute respiratory distress syndrome with autopsy. Am J Respir Crit Care Med 2013;187:761–767. Copyright ª 2013 by the American Thoracic Society

The Dynamics of Polymicrobial Biofilms To the Editor:

In their study of the respiratory microbiome, Morris and colleagues explored the microbiology of the lower respiratory tract in relation to the oral microbiome (1). Although microbial inhabitants in lung parenchyma did not appear to be entirely

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derived from direct translocation from microbial communities in the mouth as discussed, the influence and dynamics of microbial biofilms within the oral cavity, and subsequently through to the lower respiratory tract, were not discussed. The use of molecular techniques for microbial profiling has allowed the exploration of previously uncultivable organisms, which ultimately allows observation of patterns and similarities between communities of bacteria (2). Although microbiologists typically work with bacteria in planktonic culture, in biological systems, most bacteria exist in biofilms, which are complex polymicrobial networks embedded in extracellular polymeric substances to structurally enhance the biofilm (3). A biofilm is highly dynamic in terms of both behavior and microbial content, and as typified by dental plaque in the oral cavity, can play a key role in disease. Indeed, plaque-mediated infections such as dental caries and periodontal diseases actually occur when there is a “microbial shift” in the microbial community of the biofilm associated with local environment changes. Given disease associations and ease of access, oral biofilms have been extensively studied compared with those from other body sites; age, diet, smoking, health status, host defenses, pH, and the location of the biofilm itself can all contribute to biofilm modifications and adaptations. Gene expression and quorumsensing mechanisms within biofilms also support their ability to frequently adapt to differing conditions and alter their virulence (4). Although there is anatomical contiguity between the oral cavity and the distal air spaces of the lung, the species identified along the tract may not result from simple kinetics alone. Indeed, given our understanding of the dynamics of plaque biofilms, it would be surprising to observe the same distribution of bacteria in environments that differ substantially with respect to gas flow, nutrients, redox potential, and host defense molecules—even if starting with identical polymicrobial communities. In this context, the observation by Morris and colleagues that the pulmonary microbiome of healthy individuals resembles that of the oral cavity, but that the relative representation of bacterial species differs, might indeed be expected. Nevertheless, by characterizing the lung microbiome in a large cohort of healthy individuals, this study will prove invaluable for the further study of the pulmonary microbiome in respiratory diseases. Author disclosures are available with the text of this letter at www.atsjournals.org.

Kirsty Sands, B.Sc. David W. Williams, Ph.D. Melanie Wilson, Ph.D. Michael Lewis, Ph.D., B.D.S. Cardiff University Cardiff, United Kingdom Matthew P. Wise, D.Phil. University Hospital of Wales Cardiff, United Kingdom

References 1. Morris A, Beck JM, Schloss PD, Campbell TB, Crothers K, Curtis JL, Flores SC, Fontenot AP, Ghedin E, Huang L, et al.; Lung HIV Microbiome Project. Comparison of the respiratory microbiome in healthy nonsmokers and smokers. Am J Respir Crit Care Med 2013;187: 1067–1075. 2. Dewhirst FE, Chen T, Izard J, Paster BJ, Tanner ACR, Yu W-H, Lakshmanan A, Wade WG. The human oral microbiome. J Bacteriol 2010;192:5002–5017. 3. Avila M, Ojcius DM, Yilmaz O. The oral microbiota: living with a permanent guest. DNA Cell Biol 2009;28:405–411. 4. Sifri CD. Healthcare epidemiology: quorum sensing: bacteria talk sense. Clin Infect Dis 2008;47:1070–1076. Copyright ª 2013 by the American Thoracic Society

Reply: Moving forward in sepsis research.

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