Mechanical ventilation is a vital intervention in the management of patients who need general anesthesia for surgery. Ventilation is increasingly recognized for its effects on pulmonary integrity, also known as ventilator-associated lung injury in patients, and ventilator-induced lung injury (VILI) in animal models of ventilation (1–3).
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 inflammation. 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 upregulation 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 (7–9). 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 ventilated 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.
MATERIALS AND METHODS
TLR2 mRNA expression in ventilated patients and mice
This is a secondary analysis of samples obtained from a previous randomized controlled trial of ventilation in patients under general anesthesia for surgery without preexisting lung injury (10). The institutional review board of the Academic Medical Center (AMC), Amsterdam, the Netherlands, approved the study protocol, and informed consent was obtained from all patients before study entry.
The study protocol was described in detail previously (10). In short, patients were randomized to ventilation with a conventional tidal volume (VT) of 12 mL/kg predicted body weight without positive end-expiratory pressure (PEEP) or to ventilation with a protective VT of 6 mL/kg predicted body weight with a PEEP level of 10 cmH2O. Bronchoalveolar lavage fluid was obtained twice, directly after initiation of ventilation in the right middle lobe or lingula and after 5 h in the contralateral lung, and processed as described previously (10).
Bronchoalveolar lavage fluid cells were used to determine TLR2 messenger ribonucleic acid (mRNA) levels relative to the housekeeping gene hypoxanthine-guanine phosphoribosyl transferase (HPRT) as described previously (11). The following human primer sequences were used: TLR2 forward primer 5’-catgtgcgtggccagcaggt-3’ and reverse primer 5’-cccccgtgagcaggatcagc-3’and HPRT forward primer: 5’-tgctgacctgctggattaca-3’ and reverse primer 5’-cctgaccaaggaaagcaaag-3’. Analysis included samples in which paired measurement of both time points was possible. Murine pulmonary TLR2 and TLR4 expression was determined in lung homogenate samples obtained from mice ventilated for 5 hours with lower VT (7.5 ml/kg) or higher VT (15 ml/kg) (n = 7/group), non-ventilated mice served as controls (n = 4/group) (11). Messenger RNA analysis was performed as previously described (11). The following murine primer sequences were used: TLR2 forward primer 5′-tctgggcagtcttgaacattt-3′ and reverse primer 5′-agagtcaggtgatggatgtcg3′, TLR4 forward primer 5′-ggactctgatcatggcactg-3′ and reverse primer 5′-ctgatccatgcattggtaggt-3′, and HPRT forward primer 5′-tcctcctcagaccgctttt-3′ and reverse primer 5′cctggttcatcatcgctaatc-3′.
TLR2 in murine VILI
The Animal Use and Care Committee of the AMC approved the experiments. Nine-week-old C57Bl/6 mice were purchased from Harlan Inc. (Horst, the Netherlands). Toll-like receptor 2 KO mice, generated as described previously (12) and backcrossed more than six times to a C57Bl/6 background, were bred in the animal facility of the AMC.
Ventilation was performed as described previously (13). In short, mice received pressure controlled ventilation for 5 h, with higher VT (∼15 mL/kg) without PEEP (HVT) or lower VT (∼7 mL/kg) with a PEEP level of 3 cmH2O (n = 7 per group). Nonventilated mice served as controls (n = 4 per group). Blood pressure and heart rate were monitored at t = 0 h, t = 2.5 h, and t = 5 h using a murine tail-cuff system. Data were recorded on a data acquisition system (PowerLab/4SP, ADInstruments, Spenbach, Germany).
Mice were sacrificed after 5 h by withdrawing blood from the carotid artery; in ventilated mice, this was used for blood gas analysis. Then, the left lung was snap frozen and homogenized, and the right lung was used to obtain BALF as described previously (13). Bronchoalveolar lavage fluid cell counts were determined using a Coulter cell counter (Beckman Coulter, Fullerton, Calif), differential cell counts were performed on Giemsa-stained cytospin preparations. Total protein levels in BALF were measured using a Bradford Protein Assay Kit (OZ Biosciences, Marseille, France). Immunoglobulin M (IgM) levels were measured as previously described (13). Interleukin 6 (IL-6), IL-1β, and keratinocyte-derived chemokine (KC) were determined in lung tissue homogenate by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minn). Hyaluronan levels were measured in BALF by enzyme-linked immunosorbent assay (R&D Systems).
Data are presented as mean (SEM). Human samples were analyzed by paired t test or Wilcoxon signed-rank test. One outlier (Grubbs test) was removed from the LVT ventilated group; removal did not affect outcome. Murine data were analyzed by one-way analysis of variance with Bonferroni post hoc test or Kruskal-Wallis test, followed by Mann-Whitney U test, depending on data distribution. Values of P < 0.05 were considered statistically significant.
TLR2 mRNA expression levels are enhanced in ventilated lungs
Patient characteristics and perioperative parameters were previously described in detail (10). Ninety-nine percent of BALF cells obtained before and after 5 h of ventilation were macrophages. Toll-like receptor 2 mRNA levels in cells obtained from patients ventilated with lower VT and PEEP were not significantly different compared with baseline (n = 17 pairs). Toll-like receptor 2 levels in patients ventilated with higher VT and no PEEP, however, were significantly upregulated after 5 h of ventilation (n = 16 pairs) (Fig. 1A). In line, ventilation enhanced the levels of TLR2 mRNA in total lung homogenates of mice, reaching significance for mice ventilated with the injurious strategy (Fig. 1B).
TLR2 deficiency aggravates VILI
Next, WT and TLR2 KO mice were ventilated to study the role of TLR2 in VILI pathogenesis. Heart rate and systolic blood pressures remained stable throughout the experiment, with no differences between WT and KO mice (Fig. 2, A and B). Blood gas analysis demonstrated adequate gas exchange in ventilated mice, with no differences between WT and TLR2 KO mice (Table 1). As a measure of alveolar-capillary membrane permeability, we analyzed total protein levels and IgM levels in BALF (Fig. 3, A and B). Mechanical ventilation did not affect total protein concentrations. Immunoglobulin M levels, however, were increased by the injurious ventilation strategy compared with nonventilated controls (P < 0.05). Further analysis of BALF showed that both ventilation strategies induced cell influx (for both, P < 0.01) as compared with a nonventilated control group (Fig. 3C). Differential cell counts revealed an increased neutrophil presence in (for both, P < 0.01) (Fig. 3D). Furthermore, we analyzed levels of the potent cytokines IL-6 and IL-1β and the chemokine KC in pulmonary tissue (Fig. 4, A – C). Mice ventilated with the injurious strategy demonstrated higher levels of IL-6 and IL-1β when compared with the lung-protective strategy (IL-6, P < 0.01; IL-1β, P < 0.05) and to spontaneously breathing mice (for both, P < 0.01). No significant increases were found in KC concentrations in lungs of WT animals because of ventilation.
In contrast to our hypothesis, TLR2-deficient mice were not protected against VILI. To our surprise, total protein and IgM tended to be higher in ventilated TLR2 KO mice compared with WT mice. Furthermore, neutrophil influx in TLR2 KO mice was enhanced, reaching significance for TLR2 KO mice ventilated with the injurious strategy. Moreover, TLR2-deficient mice ventilated with HVT displayed higher pulmonary levels of IL-6, IL-1β, and KC.
A possible explanation for our findings could be that the lack of TLR2 results in enhanced triggering of TLR4 because some DAMPs can activate both TLR2 and TLR4. In support of this suggestion, we observed increased TLR4 mRNA expression in pulmonary tissue of ventilated mice (Fig. 5A). Moreover, hyaluronan, a well-known DAMP that can activate both TLR2 and TLR4 is increased in lung lavage fluid of ventilated mice (Fig. 5B).
This study demonstrated that ventilation is associated with increased pulmonary TLR2 gene expression. Loss of TLR2, however, aggravated VILI in mice ventilated with the injurious strategy.
Two different ventilation strategies were used to study the role of TLR2: a ventilation strategy with lower VT and PEEP, currently recommended for ventilation under general anesthesia during surgery, and a strategy with conventional VT without PEEP, which has the potential to cause lung injury (14). The effect of ventilation on pulmonary TLR2 expression is uncertain because of conflicting data in the literature. A previous murine study demonstrated that 4 h of ventilation (VT of 8 mL/kg) increased pulmonary TLR2 mRNA levels (7). More recently, a 60-fold increase of TLR2 mRNA, in line with our results, was found in lungs of rabbits ventilated for 8 h with a VT of 12 mL/kg (9). In contrast, a study conducted in rats reported no effect of 4 h of ventilation on pulmonary TLR2 levels when using a VT of 6 mL/kg or 20 mL/kg (8). Our data demonstrated elevated TLR2 mRNA levels in BALF cells of patients ventilated with a conventional strategy and in murine lung tissue ventilated with the injurious strategy, which supports the work of the first two studies. In agreement with a previous murine study (7), we found no differences in ventilation-induced injury and inflammation between WT and TLR2 KO mice ventilated with LVT and PEEP. The injurious strategy, however, aggravated VILI in TLR2 KO mice, pointing at a protective role for TLR2. In line with this, a previous study reported TLR2/TLR4 double-KO mice to be more sensitive to bleomycin or hyperoxia-induced lung injury, which was associated with enhanced epithelial cell apoptosis. A basal stimulation of nuclear-factor kappa B via TLR2/4 activation was needed to protect against proapoptotic stimuli (15). The contribution of solely TLR2 herein was not studied. A different study reported that administration of the TLR2 agonist Pam3CSK aggravated ventilation-induced inflammation, indicating that stimulation of the upregulated TLR2 pathway in VILI is not beneficial (9). A second explanation for our findings could be that TLR2 is not important in VILI pathogenesis. Loss of TLR2 may lead to increased TLR4 activation because some DAMPs, like hyaluronan, can activate both TLR2 and TLR4 (15). In line with previous research, we observed increased hyaluronan levels in BALF by mechanical ventilation (5).
Our study has limitations. First, patient samples from a previous study were used to analyze TLR2 expression. During this trial, BALF cell mRNA was stored, and we were therefore able to study TLR2 mRNA levels. However, because posttranslational modifications may occur, it is also of interest to study TLR2 on a protein level. In vitro cyclic stretch of pulmonary epithelial cells has already demonstrated TLR2 upregulation on both mRNA level and protein level (9). Second, it has been described that mice are less resistant to VILI as compared with larger species (16). Moreover, it was shown that lung mechanics between mice and men differ (17). These differences may hamper extrapolation of the murine data to the human situation.
In conclusion, TLR2 is overexpressed in lungs ventilated with higher tidal volumes and no PEEP. Toll-like receptor 2 deficiency, however, did not protect lungs from VILI. Instead, inflammation induced by injurious ventilation was aggravated in TLR2 KO mice.
1. The Acute Respiratory Distress Syndrome Network. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med
342: 1301–1308, 2000.
2. Determann RM, Royakkers A, Wolthuis EK, Vlaar AP, Choi G, Paulus F, Hofstra JJ, de Graaff MJ, Korevaar JC, Schultz MJ: Ventilation with lower tidal volumes as compared with conventional tidal volumes for patients without acute lung injury: a preventive randomized controlled trial. Crit Care
14: R1, 2010.
3. dos Santos CC, Slutsky AS: The contribution of biophysical lung injury to the development of biotrauma. Annu Rev Physiol
68: 585–618, 2006.
4. Kawai T, Akira S: The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol
11: 373–384, 2010.
5. Kuipers MT, van der Poll T, Schultz MJ, Wieland CW: Bench-to-bedside review: damage-associated molecular patterns in the onset of ventilator-induced lung injury. Crit Care
15: 235, 2011.
6. Kuipers MT, Vogl T, Aslami H, Jongsma G, van den Berg E, Vlaar AP, Roelofs JJ, Juffermans NP, Schultz MJ, van der Poll T, et al.: High levels of S100A8/A9 proteins aggravate ventilator-induced lung injury via TLR4 signaling. PLoS ONE
8: e68694, 2013.
7. Vaneker M, Joosten LA, Heunks LM, Snijdelaar DG, Halbertsma FJ, van Egmond J, Netea MG, van der Hoeven JG, Schefferg GJ: Low-tidal-volume mechanical ventilation induces a toll-like receptor 4–dependent inflammatory response in healthy mice. Anesthesiology
109: 465–472, 2008.
8. Villar J, Cabrera NE, Casula M, Flores C, Valladares F, Diaz-Flores L, Muros M, Slutsky AS, Kacmarek RM: Mechanical ventilation modulates TLR4 and IRAK-3 in a non-infectious, ventilator-induced lung injury model. Respir Res
11: 27, 2010.
9. Charles PE, Tissières P, Barbar SD, Croisier D, Dufour J, Dunn-Siegrist I, Chavanet P, Pugin J: Mild-stretch mechanical ventilation upregulates toll-like receptor 2 and sensitizes the lung to bacterial lipopeptide. Crit Care
15: R181, 2011.
10. Choi G, Wolthuis EK, Bresser P, Levi M, van der Poll T, Dzoljic M, Vroom MB, Schultz MJ: Mechanical ventilation with lower tidal volumes and positive end-expiratory pressure prevents alveolar coagulation in patients without lung injury. Anesthesiology
105: 689–695, 2006.
11. Kuipers MT, Aslami H, Janczy JR, van der Sluijs KF, Vlaar AP, Wolthuis EK, Choi G, Roelofs JJ, Flavell RA, Sutterwala FS, et al.: Ventilator-induced lung injury is mediated by the NLRP3 inflammasome. Anesthesiology
116: 1104–1115, 2012.
12. Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, Takeda K, Akira S: Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity
11: 443, 1999.
13. Hegeman MA, Hemmes SN, Kuipers MT, Bos LD, Jongsma G, Roelofs JJ, van der Sluijs KF, Juffermans NP, Vroom MB, Schultz MJ: Extent of ventilator-induced lung injury in mice partly depends on duration of mechanical ventilation. Crit Care Res Pract
2013: 435236, 2013.
14. Futier E, Constantin JM, Paugam-Burtz C, Pascal J, Eurin M, Neuschwander A, Marret E, Beaussier M, Gutton C, Lefrant JY, et al.: IMPROVE Study Group. A trial of intraoperative low-tidal-volume ventilation in abdominal surgery. N Engl J Med
369: 428–437, 2013.
15. Jiang D, Liang J, Fan J, Yu S, Chen S, Luo Y, Prestwich GD, Mascarehnhas MM, Garg HG, Quinn DA, et al.: Regulation of lung injury and repair by Toll-like receptors and hyaluronan. Nat Med
11: 1173–1179, 2005.
16. Caironi P, Langer T, Carlesso E, Protti A, Gattinoni L: Time to generate ventilator-induced lung injury among mammals with healthy lungs: a unifying hypothesis. Intensive Care Medicine
37; 1913–1920, 2011.
17. Soutiere SE, Mitzner W: On defining total lung capacity in the mouse. J Appl Physiol
96: 1658–1664, 2004.
KEYWORDS/ABBREVIATIONS: TLR2; acute lung injury; mechanical ventilation; innate immunity; pattern recognition receptors; VILI—ventilator-induced lung injury; TLR—Toll-like receptor; DAMPs—damage-associated molecular patterns; WT—wild-type; KO—knockout; BALF—bronchoalveolar lavage fluid; VT—tidal volume; PEEP—positive end-expiratory pressure; mRNA—messenger ribonucleic acid; HPRT—hypoxanthine-guanine phosphoribosyl transferase; LVT—lower tidal volumes; HVT—higher tidal volumes; IgM—immunoglobulin M; IL—interleukin; KC—keratinocyte-derived chemokine© 2014 by the Shock Society