In the present study, we tested the role of TLR4-MyD88 signaling in the development of VILI. In a mouse VILI model, HTV at 20 ml/kg led to a series of pathological changes consistent with VILI. These included increased alveolar permeability () with elevated cells and protein content in alveolar space (), significant inflammatory cell infiltration and cytokine production in lung tissues (), and activation of the proinflammatory NF-κB and MAPK-ERK pathways (). We demonstrate that TLR4 is essential in the development of VILI and that the critical role of TLR4 is most likely mediated via MyD88 signaling since MyD88−/− mice have attenuated pulmonary inflammation and were protected from increased pulmonary vascular permeability (). These results suggest that TLR4-MyD88 signaling pathway may be critical for the development of VILI.
A hallmark of acute lung injury (ALI) is structural impairment in the alveolar-capillary membrane barrier with subsequent increased pulmonary vascular permeability and inflammation. Although extensive research with numerous animal models has attempted to identify effective therapeutic strategies for ALI for almost 35 yr, at present, research on the effects of HTV mechanical ventilation has had perhaps the most significant impact on clinical practice, and mechanical ventilation with reduced tidal volume ventilation strategies is the only definitive treatment modality to improve survival in patients with ALI.3,22,23
However, emerging evidence suggests that mechanical ventilation, even at protective low tidal volume, may activate an inflammatory response in the lung and may cause or predispose to VILI.6–8,16
For example, data from computed tomography demonstrate that even current protective LTV may produce tidal hyperinflation in patients with acute respiratory distress syndrome due to its anatomical heterogeneity of damaged lung,24,25
from which VILI may be considered a regional phenomenon, and thus additional information on the effect of high (and low) tidal volume mechanical ventilation in experimental animals remains an important translational effort. Further improvements of this model may involve additional injurious stimuli to mechanical ventilation, as suggested by the importance of “two-hit” animal model of ALI that has been emphasized by the National Institutes of Health,26
to simulate comorbidities and complexities of ALI. Nonetheless, the standard VILI model that we adapted has been an important contributor to insights into VILI. It is noteworthy that we took great care to continuously monitor oxygen saturation and heart rate in the anesthetized mice. In addition, mice were supplemented with oxygen under anesthesia with the fraction of inspired oxygen less than 50% to prevent possible hypoxia while purposefully minimizing chances of hyperoxia.
Lung inflammation, induced by alveolar over-distention during injurious mechanical ventilation, is considered to contribute to VILI.27,28
Toll-like receptors have long been recognized to play a crucial role in innate immune response and adaptive immune response to pathogens and to non-infectious tissue injury.10–14
TLR4 is unique among the TLRs because it is the only known TLR able to activate both MyD88-dependent and TRIF-dependent signaling pathways.11,29
More importantly, it has been demonstrated that TLR4 is activated after mechanical ventilation with low tidal volume ventilation7
and plays a critical role in ALI induced by high tidal volume ventilation,9
and ischemia and reperfusion injury.32
It is generally considered that TLR4 induces two downstream signaling pathways: MyD88-dependent and TRIF-dependent pathways, simultaneously from the plasma membrane.13,14
Upon binding to endogenous activators, TLR4 forms a dimmer and recruits the downstream adaptor molecule MyD88, ultimately leading to the activation of NF-κB and activator protein 1, inducing transcription of proinflammatory genes, and resulting in cytokine production (). However, a recent study also showed that TLR4 may activate these two signaling pathways in a sequential manner; the MyD88 pathway is induced from the plasma membrane, whereas the TRIF pathway is induced from endosomes.33
Lately, Vaneker and colleagues16
demonstrated that lung inflammation was induced by mechanical ventilation with a low tidal volume (8 ml/kg) and that the effect is TRIF-dependent. In general, we did not detect significant pulmonary inflammation with our LTV protocol (7 ml/kg) but it is noteworthy that we instrumented (e.g.,
tracheostomy) our spontaneously breathing controls whereas Veneker et al16
used uncatheterized spontaneous breathing mice as control. As such, it is possible that the tracheostomy itself produced a sufficient inflammatory response to obscure the effect of LTV and/or to predispose the mice to injury from additional insult such as HTV mechanical ventilation. Nevertheless, LTV mechanical ventilation did not change pulmonary function such as EBA permeability, BAL cell counts and protein content as well as TLR4 downstream signaling pathways such as degradation of IκBα and MAPK/ERK phosphorylation. However, there was two-fold increase in plasma level of IL-6 with LTV compared to spontaneously tracheotomized control mice, a similar finding with recent study7
using mechanical ventilation at a tidal volume of 10 ml/kg for 6 h, indicating a relative small extent increase of systemic levels of IL-6 may not correlate to pulmonary function. In the current study, we also demonstrated that MyD88−/−
mice elicited marked attenuation in the lung injury, including pulmonary capillary leak, pulmonary protein and cell emigration, and IL-6 production following HTV mechanical ventilation. Moreover, we found that both TLR4-mutant and MyD88−/−
mice had attenuated NF-κB and MAPK/ERK activation in the lung, suggesting that the TLR4-MyD88 signaling contributes to the pro-inflammatory response during VILI. Taken together, these data suggest that pulmonary TLR4-MyD88 signaling may play a pivotal role in the pathogenesis of VILI and is responsible for the inflammatory changes observed in the injured lung.
TLR4-MyD88 signaling pathway following VILI
It is unclear how TLR4-MyD88 (and TLR4-TRIF) signaling is activated in response to VILI. Lipopolysaccharide, itself, the pathogenic ligand of TLR4 is responsible for TLR4 activation in Gram-negative bacterial infection, and might have been a contributing factor. Although its potential role was minimized in a recent study16
in which Vaneker et al
carefully monitored the presence of lipopolysaccharide, we are uncertain of any contaminating levels during our protocols. Nonetheless, all our surgical procedures were done in a sterile environment. In addition, the sham control mice underwent the same surgical intervention and were used to offset the possible effect of lipopolysaccharide in the animal group that underwent mechanical ventilation. In addition to lipopolysaccharide, recent evidence suggests an important role of endogenous ligands released in the setting of noninfectious tissue in activating TLR4. Examples of these potential endogenous ligands include high-mobility group box 1 protein released from necrotic cells, oxidized phospholipids from local generation of reactive oxygen species, low molecular weight hyaluran and fibrinogen from degraded extracellular matrix, heat shock proteins from necrotic cells, and surfactant protein-A.34–42
In light of some controversy between the detrimental effects of oxidized phospholipids via
and the protective effects of oxidized phospholipids via
intravenous route 43,44
in VILI, it may be particularly fruitful to clarify the role of oxidized phospholipids as an endogenous ligand and a potential future therapeutic agent, especially through intratracheal administration. It is possible that the extent and duration of cyclic stretch by mechanical ventilation may trigger different quantitative and qualitative oxidized phospholipids that may produce either antiinflammatory or proinflammatory effects to regulate pulmonary permeability.
In the Acute Respiratory Distress Syndrome Network study,3
plasma IL-6 levels had a positive correlation to patients’ mortality when comparing patients with ALI supported by mechanical ventilation with LTV to that with HTV. In addition, lower tidal volume ventilation was associated with a decrease in plasma IL-6 levels.45
In our study, we confirmed the clinical studies as HTV elicited significant increase in IL-6 levels in both BAL and plasma following 4 h of HTV (). The extent of IL-6 elevation correlates to increased BAL cell counts, indicating greater neutrophil migration and accumulation in the airspace. In the current study, we also found that HTV increased MIP-2 production in the bronchoalveolar lavage fluid ( and ). MIP-2 has been reported to be elevated in VILI and shown to augment migration of neutrophils into the alveoli.46
Although our data showed that LTV did not increase pulmonary permeability and pulmonary edema, LTV did not prevent increase of IL-6 in BAL and plasma, indicating that IL-6 may not be a causative factor for VILI, and increased level of IL-6 may be an adaptive host defense to potential injurious MV. Indeed in a recent study,47
IL-6 actually played a protective role in VILI and VILI is considered as a neutrophil-dependent process. The data from our group48
supports such a notion, which showed that the absence of neutrophil elastase potentially resulted in greater instances of VILI.
In the current study, we did not try to distinguish between the magnitude of contributions of downstream pathways of TLR4 (e.g.,
MyD88- or TRIF-dependent) signaling in VILI, as previous studies16
have also shown the role of TLR4-TRIF pathway in the development of inflammatory response in VILI. The strain of mouse used is a major confounding variable to contrast studies performed in different laboratories and we have systematically described this with respect to VILI.*
In this regard, C57BL/6J are relatively resistant to HTV-induced lung injury (). Nonetheless, within this strain (with MyD88 null mice being backbred on C57BL/6J background), it was apparent that MyD88−/−
mice were resistant to VILI-induced increases in permeability, histopathologic damage, and cytokine production, consistent with an important role for this limb of TLR4 signaling.
The role of gender to VILI remains unclear and may have contributed to any differences from other existing reports. We chose female mice because they have been reported to have increased sensitivity to hyperoxia49
induced acute lung injury.
No single animal model reproduces all of the characteristics of acute lung injury, and discrepancies exist between effective therapeutic approaches in animal studies and unsuccessful therapies in human clinical trials. Small animals, such as mice, are a very powerful research tool because they can be genetically modified to facilitate the detailed mechanistic study of complex pathways. It is technically challenging, however, to reproduce long-term mechanical ventilation in mice52
(compared to human patients) and thus in this regard, results from most animal studies need to be interpreted with caution. Nonetheless, short-term mechanical ventilation in rodents remains the most common model used and information derived from these studies has been useful in influencing clinical practice in humans.53,54
The mechanical ventilation model we used here combined high tidal volume ventilation and tracheotomy to reproduce clinical characteristics of acute lung injury in humans, namely increased pulmonary permeability and inflammation, although other factors may influence the magnitude of lung injury such as anesthetic use (e.g.,
variability of pressure support57
and positive end-expiratory pressure.58
The insights gained from the study may still have some important clinical implications. Although lung-protective ventilation strategies using low tidal volume with limited airway pressure and appropriate positive end-expiratory pressure has been proposed as a current recommendation for the management of critically ill patients, however, a world-wide survey has been found that it is not uncommon for critically ill patients to still be ventilated with high tidal volume.59
In addition, recent computed tomography images for patients with lung injury have demonstrated nonhomogeneous distribution of pulmonary aeration and thus normally relatively small aerated lung regions may receive the largest part of tidal volume and thereby be exposed to excessive alveolar wall tension and stress due to overdistention.25,60
From a clinical point of view, the high tidal volume mechanical ventilation mouse model is still of value in clinical relevance. The emerging evidence of clinical trials and animal models of VILI, including our current study, has shown that routine use of low tidal volume seems beneficial in all patients requiring mechanical ventilation especially for those patients with preexisting lung injury and inflammation.
In summary, our results demonstrate a critical role of TLR4 and its adaptor MyD88 in the development of VILI. The TLR4-MyD88 signaling pathway may possibly act via the mechanisms involving activation of NF-κB and MAPK. Strategies to modulate NF-κB and MAPK activation and routine use of low tidal volume mechanical ventilation may have potential therapeutic benefits in patients suffering from VILI.