TLR2 DEFICIENCY AGGRAVATES LUNG INJURY CAUSED BY MECHANICAL VENTILATION Maria Theresa Kuipers,* † Geartsje Jongsma,* Maria A. Hegeman,* Anita M. Tuip-de Boer,* Esther K. Wolthuis, ‡ Goda Choi, § Paul Bresser, || Tom van der Poll, †¶ Marcus J. Schultz,*** and Catharina W. Wieland* *Laboratory of Experimental Intensive Care and Anesthesiology (LEICA), and † Center of Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam; ‡ Department of Intensive Care, Saint Lucas Andreas Hospital; § Department of Internal Medicine, Academic Medical Center, University of Amsterdam; || Department of Respiratory Medicine, Onze Lieve Vrouwe Gasthuis; and ¶ Division of Infectious Diseases, and **Department of Intensive Care, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Received 22 Nov 2013; first review completed 10 Dec 2013; accepted in final form 7 Mar 2014 ABSTRACT—Innate immunity pathways are found to play an important role in ventilator-induced lung injury. We analyzed pulmonary expression of Toll-like receptor 2 (TLR2) in humans and mice and determined the role of TLR2 in the patho- genesis of ventilator-induced lung injury in mice. Toll-like receptor 2 gene expression was analyzed in human bronchoal- veolar lavage fluid (BALF) cells and murine lung tissue after 5 h of ventilation. In addition, wild-type (WT) and TLR2 knockout (KO) mice were ventilated with either lower tidal volumes (V T ) of 7 mL/kg with positive end-expiratory pressure (PEEP) or higher V T of 15 mL/kg without PEEP for 5 h. Spontaneously breathing mice served as controls. Total protein and immunoglobulin M levels in BALF, neutrophil influx into the alveolar compartment, and interleukin 6 (IL-6), IL-1", and keratinocyte-derived chemokine concentrations in lung tissue homogenates were measured. We observed enhanced TLR2 gene expression in BALF cells of ventilated patients and in lung tissue of ventilated mice. In WT mice, ventilation with higher V T without PEEP resulted in lung injury and inflammation with higher immunoglobulin M levels, neutrophil influx, and levels of inflammatory mediators compared with controls. In TLR2 KO mice, neutrophil influx and IL-6, IL-1", and keratinocyte- derived chemokine were enhanced by this ventilation strategy. Ventilation with lower V T with PEEP only increased neu- trophil influx and was similar in WT and TLR2 KO mice. In summary, injurious ventilation enhances TLR2 expression in lungs. Toll-like receptor 2 deficiency does not protect lungs from ventilator-induced lung injury. In contrast, ventilation with higher V T without PEEP aggravates inflammation in TLR2 KO mice. KEYWORDS—TLR2, acute lung injury, mechanical ventilation, innate immunity, pattern recognition receptors ABBREVIATIONS—VILIVventilator-induced lung injury; TLRVToll-like receptor; DAMPsVdamage-associated molecular patterns; WTVwild-type; KOVknockout; BALFVbronchoalveolar lavage fluid; V T Vtidal volume; PEEPVpositive end-expiratory pressure; mRNAVmessenger ribonucleic acid; HPRTVhypoxanthine-guanine phosphoribosyl transferase; LV T Vlower tidal volumes; HV T Vhigher tidal volumes; IgMVimmunoglobulin M; ILVinterleukin; KCVkeratinocyte-derived chemokine INTRODUCTION Mechanical ventilation is a vital intervention in the man- agement of patients who need general anesthesia for surgery. Ventilation is increasingly recognized for its effects on pulmo- nary integrity, also known as ventilator-associated lung injury in patients, and ventilator-induced lung injury (VILI) in animal models of ventilation (1Y3). The innate immune system seems to play an important role in injury and inflammation caused by ventilation (3). Toll-like receptors (TLRs) are considered key mediators of the innate immune response (4). These receptors recognize both pathogen- associated molecular patterns and damage-associated molecular patterns (DAMPs). Both molecular patterns trigger inflamma- tion. Indeed, several studies have indicated that ventilation could trigger the release of TLR2 and/or TLR4, activating DAMPs (e.g., hyaluronan, S100A8/A9, high-mobility group box 1) (5, 6). Animal experiments demonstrated ventilation-induced upreg- ulation of pulmonary TLR4 expression (7, 8). In addition, loss of TLR4 clearly attenuated the development of VILI in mice (7). The role of TLR2 is more uncertain because conflicting data are reported concerning ventilation-induced pulmonary TLR2 expression (7Y9). Four hours of lung-protective ventilation did not result in differences in lung inflammation between wild- type (WT) and TLR2 knockout (KO) mice (7). Experimental studies reported, however, that injurious ventilation releases TLR2 activating DAMPs such as hyaluronan (5). Because overstretching and repetitive opening and collapsing of alveoli are important contributing factors in the pathophysiology of VILI, it is of interest to study the role of TLR2 in both lung-protective ventilation and lung-injurious ventilation. In this present study, we investigated TLR2 expression in human bronchoalveolar lavage fluid (BALF) cells of venti- lated patients undergoing elective surgery before and after 5 h of ventilation. In addition, we studied TLR2 expression in lungs of ventilated mice. In the light of the inflammatory function of TLR4 in VILI, we hypothesized that TLR2 KO mice would be protected against mechanical ventilation-induced lung injury and inflammation induced by lung-injurious ventilation. 60 SHOCK, Vol. 42, No. 1, pp. 60Y64, 2014 Address reprint requests to Maria Theresa Kuipers, MD, Laboratory of Experimental Intensive Care and Anesthesiology (LEICA), Academic Medical Center, Room M0-220, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands. E-mail: Ilse.Kuipers@amc.nl. DOI: 10.1097/SHK.0000000000000172 Copyright Ó 2014 by the Shock Society Copyright © 2014 by the Shock Society. Unauthorized reproduction of this article is prohibited.