Mild-stretch mechanical ventilation upregulates toll-like receptor 2 and sensitizes the lung to bacterial lipopeptide

Abstract

Introduction: Mechanical ventilation (MV) could prime the lung toward an inflammatory response if exposed to another insult such as bacterial invasion. The underlying mechanisms are not so far clear. Toll-like receptors (TLRs) allow the host to recognize selectively bacterial pathogens and in turn to trigger an immune response. We therefore hypothesized that MV modulates TLR2 expression and in turn modifies responsiveness to agonists such as bacterial lipopeptide (BLP).

Method: Both in vitro and in vivo experiments were conducted. First, TLR2 expression and protein were measured in the A549 pulmonary epithelial cell line submitted to 8-hour cyclic stretch (20% elongation; 20/minute rate). After a 24-hour period of cyclic stretch, the inflammatory response of the A549 cells to the synthetic BLP, Pam3CSK4, was tested after 8 hours of exposure. In a second set of experiments, healthy anesthetized and paralyzed rabbits were submitted to 8-hour MV (tidal volume = 12 ml/kg, zero end-expiratory pressure; FIO2 = 50%; respiratory rate = 20/minute) before being sacrificed for TLR2 lung expression assessment. The lung inflammatory response to BLP was then tested in animals submitted to 24-hour MV before being sacrificed 8 hours after the tracheal instillation of Pam3CSK4.

Results: Cyclic stretch of human pulmonary epithelial cell lines increased both TLR2 mRNA and protein expression. Cells submitted to cyclic stretch also increased IL-6 and IL-8 secretion in response to Pam3CSK4, a classical TLR2 ligand. A mild-stretch MV protocol induced a 60-fold increase of TLR2 mRNA expression in lung tissue when compared with spontaneously breathing controls. Moreover, the combination of MV and airway exposure to Pam3CSK4 acted synergistically in causing lung inflammation and injury.

Conclusions: Mild-stretch MV increases lung expression of TLR2 and sensitizes the lung to bacterial TLR2 ligands. This may account for the propensity of mechanically ventilated patients to develop acute lung injury in the context of airway bacterial colonization/infection.

Figure 1

Experimental design description. (a) In vitro experiments. (b) In vivo experiments. FACS, fluorescence-activated cell sorting; MV, mechanical ventilation; qPCR, quantitative PCR; SB, spontaneously breathing; TLR, toll-like receptor.

Figure 2

Pathogen recognition receptor expression in cells submitted to cyclic stretch or kept in static condition. (a) mRNA fold induction in A549 cells stretched for 8 hours or kept in static condition. All values are shown as the fold increase compared with control static cells normalized to 1. IL-8 mRNA levels induced by stretch served as a positive control. Both toll-like receptor (TLR)2 and TLR4 mRNA levels were markedly increased by cyclic stretch. MD-2 and CD14 mRNA levels were not significantly modified by stretch. Results are expressed as mean ± standard deviation of three distinct experiments. (b) Surface expression of TLR2, TLR4, MD-2 and CD14 by fluorescence-activated cell sorting (FACS) in A549 cells submitted to a 24-hour cyclic stretch or in cells kept in static condition. Except for CD14, none of these receptors showed significant surface expression. (c) Intracellular expression of TLR2, TLR4, MD-2 and CD14 by FACS in permeabilized A549 cells submitted to a 24-hour cyclic stretch or in cells kept in static condition. TLR2 was detected in permeabilized A549 cells after 24 hours of cyclic stretch. CD14 was detected, but not increased, by cyclic stretch. Neither TLR4 nor MD-2 proteins were detected by FACS in permeabilized cells, regardless of the experimental condition. Results are expressed as the mean fluorescence index (MFI) ± standard deviation of three distinct experiments. *P < 0.05 between static and stretched cells.

Figure 3

Inflammatory mediator production by A549 cells stimulated with Pam₃CSK₄ in static and cyclic stretch conditions. (a) IL-6 and IL-8 protein concentrations in conditioned supernatants from cells stretched for 24 hours or kept in static condition, and subsequently stimulated for 8 hours with bacterial lipopeptide (BLP). Cyclic stretch increased the inflammatory response of A549 cells in response to BLP. (b) IL-6 and IL-8 mRNA levels in human type II-like A549 alveolar cells stretched for 24 hours or kept in static condition, and subsequently stimulated for 8 hours with BLP. mRNA levels were normalized to the expression of GAPDH mRNA. All values are reported as the fold increase compared with control static cells normalized to 1. For both inflammatory markers, a clear synergistic effect between cyclic stretch and BLP is observed. Results are expressed as the mean ± standard deviation of four distinct experiments. *P < 0.05 between static and stretched cells stimulated with BLP.

Figure 4

Toll-like receptor 2 expression in A549 cells stretched with various inhibitors. IL-8 mRNA levels were measured as a positive control of cell activation by cyclic stretch. mRNA levels were normalized with the expression of GAPDH mRNA. All values are reported as the fold increase compared with control static cells normalized to 1. SB203580 significantly reduced the toll-like receptor (TLR)2 mRNA level induced by stretch, whereas Sn50 had no effect. The opposite was observed for IL-8 expression induced by cyclic stretch. Results are expressed as the mean ± standard error of the mean of three distinct experiments. *P < 0.05 between stretched cells with or without SB 203580. SB, spontaneously breathing.

Figure 5

Pathogen recognition receptor expression in rabbits submitted to mechanical ventilation or kept spontaneously breathing. Lung IL-8 mRNA levels were measured as a positive control of lung inflammation induced by mechanical ventilation (MV). mRNA levels were normalized with the expression of GAPDH mRNA. All values are shown as the fold increase compared with spontaneously breathing (SB) rabbits normalized to 1. MV induced a strong increase of toll-like receptor (TLR)2 mRNA levels, higher than TLR4, MD-2 and CD14. Results are expressed as mean ± standard deviation. *P < 0.05 between SB and MV rabbits.

Figure 6

Light photomicrographs of rabbit lungs in various conditions. (a) Lung from a spontaneously breathing (SB) animal. (b) Rabbit lung after 32-hour mild-stretch mechanical ventilation (MV). (c) Lung from a SB animal following tracheal instillation of the synthetic bacterial lipopeptide (BLP), Pam₃CSK₄, and sacrificed 8 hours later. (d) Lung from an animal submitted to 24-hour mild-stretch MV followed by tracheal instillation of BLP and sacrifice 8 hours later. The main abnormalities were septal thickening and inflammatory cell infiltration in rabbits submitted to MV, increased by the presence of BLP. Representative fields, original magnification, x10; hematoxylin and eosin stain.

Figure 7

IL-8 expression following instillation of Pam₃CSK₄ in rabbits submitted previously, or not, to mechanical ventilation. IL-8 mRNA levels were normalized with the expression of GAPDH mRNA. Values are reported as the fold increase compared with control spontaneously breathing (SB) animals normalized to 1 (n = 4 per group). Results are expressed as the mean ± standard deviation. Lung response to bacterial lipopeptide (BLP) was markedly enhanced by previous mechanical ventilation (MV). *P < 0.05 between SB and MV stimulated rabbits.