In patients that experience severe trauma, undergo lung transplantation, have pulmonary emboli, or have severe sepsis, lung I/R injury can be severe enough to require mechanical ventilatory support, which in turn can contribute to overall morbidity and mortality (reviewed in 42
In this study we utilized a precise and sophisticated murine model to study isolated lung I/R injury. As opposed to the majority of published reports on lung I/R injury in which hilar clamping is employed5,6
we focus on direct lung injury from I/R alone. The contribution of atelectasis, lung collapse and mechanical ventilation to lung injury is well documented43–46
and by allowing spontaneous ventilation throughout the majority of the I/R period in our model, we are able to minimize the deletrious effects of lung collapse and mechanical ventilation. In addition we chose to study I/R injury in mice, so as to take advantage of available gene knockout and transgenic animals. Overall, we hope to understand the molecular and cellular basis of this pathophysiological response. In our mouse I/R model, we recapitulated neutrophil trafficking - a hallmark of clinical I/R injury - and this correlated well with the induction of inflammatory markers early on in the reperfusion period. IL-6 and IL-1β were found to be specifically upregulated both at the message and protein levels early in this process while TNFα, type I IFN, IL-10, IL-17, and inducible nitric oxide synthase (iNOS) were not. We observed early upregulation of specific chemokines that guide neutrophils to the site of injury (CXCL1 and 2). This appeared to be an innate immune process since IL-17, an adaptive immune T cell cytokine that results in neutrophil chemotaxis, was not induced by I/R in the lung. We were also able to detect a significant increase in HMGB1 levels in our I/R mice and this damage marker could play a key role in ventilated lung I/R injury. Future work will attempt to identify the key damage markers released and their roles in mediating I/R injury. It is likely that reactive oxygen species generation plays an important role in this process as inhibition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has been shown to reduce I/R injury in this model47
Examining mice that were defective or deficient for TLR4 signaling revealed an important role for TLR4 in lung I/R injury. In contrast, TLR2 −/− mice did not demonstrate similar levels of resistance to I/R injury. Our results are consistent with other published data that implicate TLR4 as a key receptor in lung I/R injury. However, these prior studies employed hilar clamping and thus did not focus on I/R injury alone because the atelectasis associated with occluding the left main bronchus along with the left pulmonary artery likely generated an additional source of inflammation which could also be TLR4-dependent5,6
. We focused specifically on ventilated I/R injury and showed that TLR4 signaling was involved early in lung I/R injury. We hypothesize that TLR4 directly or indirectly senses damage patterns released by EC in reperfused vasculature.
EC express TLR4 and TLR2. Specifically, EC can respond to both TLR4 and TLR2 ligands to produce IL-6. As demonstrated by our in vitro
data, simulated nutritional I/R injury in the absence of shear stress changes that accompany flow alterations generated EC IL-6, but not IL-1β production soon after reperfusion (). The lack of IL-1β production by EC led us to create co-culture systems of EC with macrophages to identify the cell type producing IL-1β. We hypothesized that IL-1β made by macrophages acted on EC, amplifying IL-6 produciton under nutritional I/R conditions (). In studies examining EC responses to lipopolysaccharide (LPS) in the presence of PBMC monocytes, Sabroe and colleagues showed that IL-1β production fed back on EC to augment inflammatory cytokine production48
. We suggest that this paradigm of monocytic cell types serving as sensors/amplifiers of pathogen-mediated inflammatory responses may apply to conditions of sterile inflammation in the presence of damage markers.
Macrophages have been implicated in some I/R injury models of the heart, liver, and even lung (in the setting of lung transplantation)19,49–53
. To assess the in vivo
role of macrophages in the lung I/R mediated inflammatory process, we used liposome encapsulated clodronate to deplete all macrophage cells in the mouse38
and demonstrated a failure in neutrophil recruitment following lung I/R. Liposome encapsulated clodronate treatment eliminates macrophage populations accessible to the blood stream (splenic macrophages, for example) and can also eliminate resident tissue macrophage populations (such as AM and interstitial lung macrophages) by targeting circulating blood monocyte precursors38,40,54,55
AM are a major subset of macrophages that reside in the lung. AM make up 80+% of phagocytic cells in the alveoli and are important for responding to inhaled particles and pathogens56
. In this study, we show that AM play a key role in the response to lung I/R injury. AM depletion with DTx in CD11c-DTR mice resulted in a near-complete absence of neutrophil trafficking to the lung following I/R (). Our data suggest that these cells, which do not reside inside the pulmonary vasculature, may participate in the sensing of I/R and communicate with EC leading to the production of inflammatory mediators. Although lung EC and AM may not physically be in contact with each other, their close proximity would allow them to communicate via secreted factors such as IL-1β. Furthermore, AM may perform this I/R sensing role possibly through TLR4-mediated uptake of markers released by I/R-damaged EC or epithelial cells that diffuse into the alveoli.
While reports from others have suggested a role for monocytic cells in the process of neutrophil infiltration in a murine lung transplant model19
, our data using a more focused and precise lung I/R mouse model show that a specific subset – AM – likely act as the primary sensor cell type that responds to I/R injury. One group reported results contradictary to ours with worsening of lung I/R injury with AM depletion57
. However, their rat model involved ex vivo
mechanical perfusion of isolated lungs and measurement of intrapulmonary neutrophil accumulation. In contrast, in our model we did not observe neutrophil trafficking at their early (1 h) reperfusion times (See Supplemental Digital Content 1, figure 1B
, which examines histological lung sections 1 h after reperfusion).
Two other groups demonstrated a protective effect of AM-depletion in lung I/R injury. The first also used an ex-vivo
model to study the role of AM in lung I/R injury58
. Mice were ex-sanguinated and reperfused with a buffered solution to mimic mixed venous blood. The second used hilar clamping in a rat model of non-ventilated I/R injury59
. We believe that our in vivo
model better and more closely replicates clinical I/R scenarios. Furthermore, having validated this model in mice, we can further identify and dissect the key pathways important in lung I/R injury using available genetic knockouts and transgenic mice. Through in vivo
cell depletion and reconstitution experiments, future experiments can examine the role of AM and specific signaling pathways within AM in this physiologically relevant model of ventilated lung I/R.
Two of the studies referred to previously also used liposome encapsulated clodronate to deplete AM and examined lung vascular permeability58,59
. However, liposome encapsulated clodronate may affect lung vascular permeability independent of other treatments or procedures perhaps making this AM-depletion method unsuitable for examining changes in vascular permeability.
AM frequency in the lung, their location close to the vasculature, and reported functionality in consuming dead cells and debris, arguably make them ideally suited to perform the role of sterile damage or I/R sensor. However, some published reports have characterized AM as being immunosuppressive rather than proinflammatory54,57,60
. Experiments are currently ongoing to directly address whether alveolar macrophages are necessary and sufficient for the generation of a full I/R inflammatory response. However, at this time we cannot formally rule out the possibility that another phagocytic CD11c+ population, such as a dendritic cell population, may contribute to initiating the response to lung I/R injury. It is also entirely possible that both dendritic cells and AM are immunomodulatory in distinct ways depending on the type of sterile insult, the branch of immune system activated (adaptive versus innate), and the co-presence of pathogen.
In summary, this study employs a murine model of lung injury that does not involve airway collapse and specifically focuses on ventilated I/R by isolating the blood flow to a single lung. The data provide compelling evidence that AM serve the role of sensing and/or amplifying lung I/R injury and that TLR4 plays a critical part in this process. Manipulating the activity and presence of TLR4 and AM could thus potentially permit control of the clinical response to lung I/R injury. In the future, one could envision this role of manipulating or modulating the immune system and its inflammatory responses falling to the anesthesiologist or perioperative physician in rapidly evolving clinical situations encountered in the operating theater and intensive care unit.