Atelectasis causes alveolar hypoxia-induced inflammation during uneven mechanical ventilation in rats

Abstract

Background: Patients with acute respiratory distress syndrome receiving mechanical ventilation show inhomogeneous lung aeration. Atelectasis during uneven mechanical ventilation leads to alveolar hypoxia and could therefore result in lung inflammation and injury. We aimed to elucidate whether and how atelectasis causes alveolar hypoxia-induced inflammation during uneven mechanical ventilation in an open-chest differential-ventilation rat model.

Methods: We first investigated inflammatory and histological changes in the bilateral lungs of unilaterally ventilated rats, in which the right lung was atelectatic and the left lung was ventilated with high tidal volume (HTV). In the next series, we investigated the effects of normal tidal volume (NTV) ventilation of the right lungs with 60 % O2 or 100 % N2 during HTV ventilation of the left lungs. Then, proinflammatory cytokine secretions were quantified from murine lung epithelial (MLE15) and murine alveolar macrophage (MH-S) cells cultured under a hypoxic condition (5 % O2) mimicking atelectasis. Further, activities of nuclear factor (NF)-κB and hypoxia-inducible factor (HIF)-1 were assessed in the nonventilated atelectatic lung and MLE15 cells cultured under the hypoxic condition. Finally, effects of NF-κB inhibition and HIF-1α knockdown on the cytokine secretions from MLE15 cells cultured under the hypoxic condition were assessed.

Results: The nonventilated atelectatic lungs showed inflammatory responses and minimal histological changes comparable to those of the HTV-ventilated lungs. NTV ventilation with 60 % O2 attenuated the increase in chemokine (C-X-C motif) ligand (CXCL)-1 secretion and neutrophil accumulation observed in the atelectatic lungs, but that with 100 % N2 did not. MLE15 cells cultured with tumor necrosis factor (TNF)-α under the hypoxic condition showed increased CXCL-1 secretion. NF-κB and HIF-1α were activated in the nonventilated atelectatic lungs and MLE15 cells cultured under the hypoxic condition. NF-κB inhibition abolished the hypoxia-induced increase in CXCL-1 secretion from MLE15 cells, while HIF-1α knockdown augmented it.

Conclusions: Atelectasis causes alveolar hypoxia-induced inflammatory responses including NF-κB-dependent CXCL-1 secretion from lung epithelial cells. HIF-1 activation in lung epithelial cells is an anti-inflammatory response to alveolar hypoxia in atelectatic lungs.

Fig. 1

Mean arterial pressure, peak airway pressure, and arterial blood gas analysis. a Mean arterial pressure, b peak airway pressure, c PaO₂, and d PaCO₂. *p < 0.05 compared with the BLV group (n = 8 rats per group). PaO ₂ arterial partial pressure of oxygen, PaCO ₂ arterial partial pressure of carbon dioxide, BLV bilateral lung ventilation, ULV unilateral lung ventilation

Fig. 2

Concentrations of proinflammatory cytokines and MPO in the right (atelectatic) and the left (ventilated with high tidal volume) lung homogenates. a TNF-α, b CXCL-1, c CCL-2, and d MPO concentrations were measured by ELISA and normalized to the total protein concentration. *p < 0.05 compared with the BLV group, †p < 0.05 compared with the right lungs (n = 8 rats per group). TNF-α tumor necrosis factor α, CXCL-1 chemokine (C-X-C motif) ligand 1, CCL-2 chemokine (C-C motif) ligand 2, MPO myeloperoxidase

Fig. 3

Histological assessment of lung sections after 8 h of ventilation. a–e Representative images of lung sections stained with hematoxylin and eosin. a Right and b left lung sections from the BLV group and c right (atelectatic) and d left (ventilated with high tidal volume) lung sections from the ULV group. e High-magnification images of perivascular edema from right lung sections of the ULV group. Arrows indicate neutrophils adhering to tunica intima and infiltrating to the perivascular edema. Scale bar = 200 μm. f Histological scores. BLV bilateral lung ventilation, ULV unilateral lung ventilation

Fig. 4

Activities of NF-κB and HIF-1 in right lungs a NF-κB p65 binding activities and b HIF-1α protein concentrations in right lung homogenates quantified by ELISA. c VEGFA and d GLUT1 mRNA levels in right lung homogenates measured by qPCR. *p < 0.05 compared with the BLV group (n = 8 samples per group). NF-κB nuclear factor-κB, HIF-1 hypoxia-inducible factor 1, VEGFA vascular endothelial growth factor A, GLUT1 glucose transporter-1, BLV bilateral lung ventilation, ULV unilateral lung ventilation

Fig. 5

Concentrations of proinflammatory cytokines and MPO and activity of NF-κB and HIF-1α in right lung homogenates. a TNF-α, b CXCL-1, and c MPO concentrations and d NF-κB p65 binding activities and e HIF-1α concentrations in right lung homogenates were measured by ELISA and normalized to the total protein concentration. *p < 0.05 compared with the ULV group (n = 5 rats per group). TNF-α tumor necrosis factor α, CXCL-1 chemokine (C-X-C motif) ligand 1, MPO myeloperoxidase, NF-κB nuclear factor-κB, HIF-1 hypoxia-inducible factor 1, ULV unilateral lung ventilation, NTV normal tidal volume

Fig. 6

Effect of hypoxia on cytokine secretion from MLE15 and MH-S cells and activities of NF-κB and HIF-1α in MLE15 cells. a, b Cells were cultured in 21 % O₂ (white bar) or 5 % O₂ (black bar) with or without TNF-α (500 pg/mL). a CXCL-1 secreted from MLE15 cells and b CCL-2 secreted from MH-S cells. (n = 6 samples per group). c–f MLE15 cells were cultured in 21 % O₂ (white bar) or 5 % O₂ (black bar) with TNF-α (500 pg/mL). c NF-κB p65 binding activities (n = 5 samples per group) and d HIF-1α concentrations in MLE15 cells (n = 4 samples per group). e VEGFA and f GLUT1 mRNA levels in MLE15 cells (n = 5 samples per group). *p < 0.05 compared with the 21 % O₂ group, †p < 0.05 compared with the group without TNF-α. TNF-α tumor necrosis factor α, CXCL-1 chemokine (C-X-C motif) ligand 1, CCL-2 chemokine (C-C motif) ligand 2, NF-κB nuclear factor-κB, HIF-1 hypoxia-inducible factor 1, VEGFA vascular endothelial growth factor A, GLUT1 glucose transporter-1

Fig. 7

Effects of NF-κB inhibition and HIF-1α knockdown on cytokine secretion from MLE15 cells. Cells were cultured in 21 % O₂ (white bar) or 5 % O₂ (black bar) with TNF-α (500 pg/mL). a CXCL-1 secretion from MLE15 cells cultured with 500 pg/mL TNF-α after treatment with Bay 11–7082 or vehicle. *p < 0.05 compared with the 21 % O₂ group (n = 6 samples per group). b HIF1A, VEGFA, and GLUT1 mRNA levels and c HIF-1α protein concentrations in MLE15 cells transfected with HIF-1α or nontarget siRNA. *p < 0.05 compared with the nontarget groups (n = 5 samples per group). d CXCL-1 secretion from MLE15 cells cultured with 500 pg/mL TNF-α after transfection with HIF-1α or nontarget siRNA. *p < 0.05 compared with the 21 % O₂ group, †p < 0.05 compared with the nontarget group (n = 6 samples per group). NF-κB nuclear factor-κB, HIF-1 hypoxia-inducible factor 1, VEGFA vascular endothelial growth factor A, GLUT1 glucose transporter-1, K/D knockdown, CXCL-1 chemokine (C-X-C motif) ligand 1