3D Quantitative Imaging of Unprocessed Live Tissue Reveals Epithelial Defense against Bacterial Adhesion and Subsequent Traversal Requires MyD88 Connie Tam1, Jeffrey LeDue1, James J. Mun2, Paul Herzmark3, Ellen A. Robey3, David J. Evans1,4, Suzanne M. J. Fleiszig1,2,5* 1 School of Optometry, University of California, Berkeley, California, United States of America, 2 Program in Vision Science, University of California, Berkeley, California, United States of America, 3 Department of Molecular and Cell Biology, University of California, Berkeley, California, United States of America, 4 College of Pharmacy, Touro University California, Vallejo, California, United States of America, 5 Programs in Infectious Diseases and Immunity and Microbiology, University of California, Berkeley, California, United States of America Abstract While a plethora of in vivo models exist for studying infectious disease and its resolution, few enable factors involved in the maintenance of health to be studied in situ. This is due in part to a paucity of tools for studying subtleties of bacterial-host interactions at a cellular level within live organs or tissues, requiring investigators to rely on overt outcomes (e.g. pathology) in their research. Here, a suite of imaging technologies were combined to enable 3D and temporal subcellular localization and quantification of bacterial distribution within the murine cornea without the need for tissue processing or dissection. These methods were then used to demonstrate the importance of MyD88, a central adaptor protein for Toll-Like Receptor (TLR) mediated signaling, in protecting a multilayered epithelium against both adhesion and traversal by the opportunistic bacterial pathogen Pseudomonas aeruginosa ex vivo and in vivo. Citation: Tam C, LeDue J, Mun JJ, Herzmark P, Robey EA, et al. (2011) 3D Quantitative Imaging of Unprocessed Live Tissue Reveals Epithelial Defense against Bacterial Adhesion and Subsequent Traversal Requires MyD88. PLoS ONE 6(8): e24008. doi:10.1371/journal.pone.0024008 Editor: Sunil K. Ahuja, South Texas Veterans Health Care System, United States of America Received December 9, 2010; Accepted August 3, 2011; Published August 25, 2011 Copyright: 2011 Tam 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) EY011221 and AI079192 (SMJF), AI065831 (ER) and JL was supported by EY003176. 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: fleiszig@berkeley.edu Introduction Many of our tissue surfaces are lined with epithelia that are thought to be a first line defense against infection. Yet the mechanims involved in epithelial barrier function against micro- bial traversal in vivo remain poorly understood, in part due to the paucity of suitable in vivo models. Attempts to understand factors that modulate microbe traversal of epithelia have been limited to in vitro experiments, most utilizing monolayers of cultured cells grown on permeable filters, and with an emphasis on establishing contributing virulence factors rather than determining epithelial defenses against it [1–3]. While it is known that epithelial cells do express factors with the potential to be defensive [4,5], those that actually participate in limiting bacterial trafficking through cell multilayers are yet to be determined. Moreover, relevance of in vitro studies using epithelial cells grown in culture to actual in vivo barrier function of the epithelium is not yet certain. There have been two major obstacles to the development of in vivo models to study tissue traversal by microbes and the defenses that protect against it. One is that ‘‘infection’’ models used by most investigators are disease models in which early interactions between bacteria and the epithelium are deliberately by-passed to induce disease. Understanding factors that maintain health at the surface of epithelial-lined tissues, such as those that defend against microbial traversal, require different models, and the use of outcome measures more subtle than scoring morbidity/mortality or quantification of total microbe load within the tissue. Related to this, the other major obstacle has been a lack of techniques for accurately localizing bacteria within live in vivo infected tissue. Fixation and sectioning of tissue to view microbe penetration in cross-section enables viewing of only a small area, introducing the possibility that non-representative areas are studied, or conversely, that rare but important features are missed. Other disadvantages of fixation are that it removes the opportunity to gain temporal information, and that it can impact morphology or introduce artifacts such that microbial location may be misinterpreted. The healthy corneal epithelium at the surface of the eye usually resists bacterial adhesion, which is thought to involve the presence of surface-associated mucins [6,7]. Recently we developed a means to enable bacteria to adhere to the epithelium of mouse corneas that also allowed the epithelium’s resistance to subsequent traversal by the adherent bacteria to be manipulated. This involved blotting the epithelial surface with a lint-free tissue prior to bacterial inoculation to enable bacterial adhesion. EGTA (Ethylene Glycol Tetraacetic Acid) treatment after this blotting process can then be used to toggle on epithelial susceptibility to traversal by adhering bacteria. Since EGTA is removed (by washing) prior to bacterial inoculation, the impact of this chemical treatment on traversal is likely to involve its effects on the corneal epithelium rather than on the bacteria. EGTA, a calcium-chelator, is capable of disrupting calcium dependent cell-cell junctions [8]. However, calcium is also required for many other cell functions, PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e24008