Mechanical ventilation modulates Toll-like receptors 2, 4, and 9 on alveolar macrophages in a ventilator-induced lung injury model

Abstract

Objective: To investigate the role of Toll-like receptor 2 (TLR2), TLR4, TLR9 and myeloid differentiation factor 88 (MyD88) on alveolar macrophages in ventilator-induced lung injury (VILI).

Methods: Male, adult pathogen-free Sprague-Dawley rats weighing 300-350 g were used in this study. Animals were tracheotomized and allowed to breathe spontaneously for 4 h or mechanically ventilated for 4 h with low or high tidal volume (7 or 40 mL/kg). TLR2, TLR4, and TLR9, MyD-88 and NF-κΒ of alveolar macrophages' expression under the different ventilation conditions were detected. Pulmonary permeability, lung inflammatory, IL-6 and IL-1β were assessed as well.

Results: Rats subjected to high tidal volume showed significantly greater pulmonary permeability and lung inflammatory than the control rats. Alveolar macrophages from rats subjected to high tidal volume also showed significantly higher protein expression of TLR2 (0.59±0.049 vs. 0.35±0.036 and 0.36±0.031, both P<0.001), TLR4 (0.845±0.0395 vs. 0.401±0.026 and 0.403±0.020, both P<0.001), TLR9 (0.727±0.074 vs. 0.383±0.039 and 0.367±0.043, both P<0.001), MyD-88 (1.01±0.060 vs. 0.485±0.045 and 0.507±0.046, both P<0.001) and NF-κΒ (0.776±0.067 vs. 0.448±0.043 and 0.481±0.047, both P<0.001), as well as significantly higher concentrations of IL-6 (7.32±0.24 vs. 2.42±0.13 and 2.44±0.32, both P<0.001) and IL-1β (139.95±9.37 vs. 53.63±5.26 and 53.55±6.63, both P<0.001) than the control and low tidal volume group.

Conclusions: The overexpression of TLR2, TLR4, and TLR9 on alveolar macrophages and release of pro-inflammatory cytokines play a role in VILI.

Keywords: Toll like receptor (TLR); alveolar macrophages; myeloid differentiation factor 88 (MyD88); ventilator-induced lung injury (VILI).

Figure 1

Lung tissue histopathology in spontaneously breathing control rats (A) and rats mechanically ventilated with low (B) or high (C) tidal volume. Sections were stained with hematoxylin-and-eosin. Tissue ventilated with high tidal volume showed much more inflammatory cell infiltration, alveolar 2 septal thickening, and pulmonary edema than did tissue in the other two groups.

Figure 2

Alveolar macrophage cell counts. Male rats were subjected to non-ventilate or low V_T or high V_T. After 4 h of the procedure, AM cells were isolated from the lung. Numbers of viable cells were determined by trypan blue staining and counted by a hemocytometer. Data are mean ± SD, n=10. ^#, P<0.001 vs. non-ventilated animals; ^$, P<0.001 vs. low V_T; *, P>0.05 vs. non-ventilated animals.

Figure 3

Fold changes in TLR2, TLR4, TLR9, MyD88 and NF-κB mRNA levels in alveolar macrophages of healthy rats after 4 h of spontaneous breathing (non-ventilated) or 4 h of mechanical ventilation with tidal volumes of 7 mL/kg (low V_T) or 40 mL/kg (high V_T) are shown in (B). Data are mean ± SD, n=10. Results were normalized to the values in GAPDH control amplifications, and expressed as fold changes relative to the non-ventilated control animals. *, P<0.001 vs. non-ventilated animals. TLR, Toll-like receptor; MyD88, myeloid differentiation factor 88; NF-κB, nuclear factor-κB.

Figure 4

Changes in TLR2, TLR4, TLR9, MyD88 and NF-κB protein levels on alveolar macrophages of healthy rats after 4 h of spontaneous breathing (non-ventilated) or 4 h of mechanical ventilation with tidal volumes of 7 mL/kg (low V_T) or 40 mL/kg (high V_T). Representative Western blot results are shown above, and quantitation of TLR2, TLR4, TLR9, MyD88 and NF-κB protein levels independent experiments is shown in (Figure 3A). Data are mean ± SD, n=10. Results were normalized to levels of GADPH protein detected on the same blots. *, P<0.001 vs. non-ventilated animals; ^#, P<0.001 vs. animals ventilated with low tidal volume; ^&, P>0.05 vs. non-ventilated animals. TLR, Toll-like receptor; MyD88, myeloid differentiation factor 88; NF-κB, nuclear factor-κB.