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Deciphering the Dual Nature of Toll-like Receptor 4 in Lung Ischemia Reperfusion Injury
H. E. Merry, P. S. Wolf, A. S. McCourtie, A. S. Farivar, M. S. Mulligan*. University of Washington, Seattle, WA,
Background: Toll like receptors represent a family of receptors that are the cornerstone of innate immunity. Toll-like receptor-4 (TLR-4) is perhaps the most studied from this family. Originally recognized for its role in recognition of LPS and initial role in defense against bacterial pathogens, in more recent years it has been shown to have a role in a more diverse array of disease processes, including modulation of myocardial and hepatic ischemia and reperfusion, and non-ischemic acute lung injury. Additionally TLR-4 has been shown to be upregulated in human lung transplantation. However its functional role has not been elucidated. In studies using knock-out mice, TLR-4 clearly seems to be involved in propagation of injury after ischemia and reperfusion. Conversely, activation of TLR-4 receptors prior to ischemia and reperfusion (IR) also confers protection in a variety of models of IR injury. The ability of one receptor to have dichotomous roles likely relates to the ability of TLR-4 to differentially recruit adapter proteins based on its status prior to ischemia ( i.e. naïve or exposed to a preconditioning stimulus). These studies seek to resolve how TLR-4 both promotes and attenuates injury in the lung and in specific key cells in response to ischemia-reperfusion and bacterial preconditioning.
Methods: Using a rat model of lung ischemia reperfusion injury, experimental animals were pretreated with TLR-4 siRNA to induce TLR-4 depletion prior to ischemia-reperfusion, additional animals were pretreated with LPS to induce. Lungs were then analyzed for parameters of injury, such as vascular permeability, inflammatory cell infiltration and cytokine production. Addition lungs were analyzed for TLR-4 and adapter protein expression, as well as downstream proinflammatory activation.
Additionally, primary cultures of alveolar macrophages were pretreated with TLR-4 siRNA and LPS prior to exposure to hypoxia and reoxygenation. They were then analyzed for TLR-4 and adapter protein expression, as well as for cytokine and chemokine production.
Results: Efficacy of TLR-4 depletion was confirmed by Western blot analysis of TLR-4. TLR-4 depletion with siRNA resulted in a significant (p<0.001) reduction in vascular permeability and proinflammatory signaling such as MAPK activation. Additionally, TLR-4 depletion in the alveolar macrophage led to a marked (>90%, p<0.001) reduction in proinflammatory cytokine (TNF-α and CINC) production and attenuation of proinflammatory SAPK activation (>90% reduction, p<0.001).
TLR-4 preactivation with LPS was also associated with significant protection and attenuation of downstream pro-inflammatory signaling. With TLR-4 preactivation there was differential adapter protein upregulation.
Conclusions: In lung ischemia reperfusion injury TLR-4 has a dual role in propagating inflammatory signaling and injury and in conferring protection if prestimulated. TLR-4 activation in the alveolar macrophage appears to be the key point in initiation of a pro-inflammatory response leading to lung injury. However pre-stimulation with LPS leads to differential adapter protein recruitment an attenuation of downstream inflammatory signaling thus conferring protection in the lung. These findings have significant implications for clinical lung transplantation wherein the majority of cadaveric donors have bacterial colonization of their airways. Rather than suggesting caution, this fact could signify a protective state of TLR-4 activation.
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