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Differential Toll-Like Receptor Preconditioning Responses in Lung Ischemia Reperfusion Injury
P. S. Wolf, J. Keech, H. Merry, M. Mulligan*. University of Washington, Seattle, WA,
BACKGROUND:
Lung ischemia-reperfusion injury (LIRI) is a significant clinical problem in the lung transplant population, increasing the risk for both early graft dysfunction and late graft failure. The alveolar macrophage (AM) is the centrally important cell type in coordinating the response to oxidative stress. The inflammatory responses of other constituent cell populations within the lung, such as pulmonary endothelial and epithelial cells, are necessary for full injury to manifest. Innate immune receptor activation has been implicated as an initiating event in other models of IRI. Modulation of immune receptor activation would appear attractive. Studies have suggested that preconditioning of innate immune receptors, such as TLR-4, may confer protection from subsequent oxidative stress. As most work has focused on TLR-4 signaling in monocytes/macrophages, the AM is a candidate cell type that utilizes TLR-4 dependent signaling in LIRI. Further studies of cardiac and renal IRI have indicated important roles for TLR-2 signaling and suggested endothelial cells as important sites of activation. Identifying differential TLR preconditioning responses to lung ischemia reperfusion may indicate cell specific sites of activation and provide potential therapeutic targets aimed at reducing LIRI severity.
METHODS:
Long-Evans rats weighing 280-320 grams were subjected to left lung hilar occlusion for a period of 90 minutes followed by up to four hours of reperfusion. Experimental animals were pre-treated 24 hours prior to ischemia with either the TLR-4 agonist LPS or the TLR-2 agonist Pam(3)Cys. Injury was assessed by measuring lung vascular permeability, myeloperoxidase content, and BAL cell count analysis. BAL effluent was analyzed for soluble cytokine and chemokine content via sandwich ELISA. Western blotting was performed to assess for MAPK phosphorylation patterns.
RESULTS:
LPS treated animals demonstrated significant reductions in vascular permeability, BAL cytokine content, tissue inflammatory cell infiltration and myeloperoxidase content. Pam(3)Cys treatment also conferred significant protection, but not to the degree of LPS. TLR-4 preconditioning reduced SAPK, but not ERK1/2, activation, while TLR-2 preconditioning correlated with a reduction in ERK1/2 phosphorylation, but not SAPK phosphorylation.
CONCLUSIONS:
Both TLR-4 and TLR-2 preconditioning provided significant protection from subsequent reperfusion injury. This appears to occur through modulation of differential inflammatory signaling cascades in AM (TLR-4) versus non-AM cell types (TLR-2). The relatively greater protection provided by TLR-4 preconditioning highlights the central role of the AM in LIRI generation. Understanding discrete patterns of TLR-4 versus TLR-2 activation may allow targeted, cell-specific therapeutic options in improving the severity of lung reperfusion injury.
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