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Differential Toll-like Receptor Activation in Lung Ischemia Reperfusion Injury
J. Keech, P. Wolf, H. Merry, M. Mulligan*. University of Washington, Seattle, WA,
BACKGROUND: The objective of our study was to determine differential TLR2 and TLR4 dependent proinflammatory signaling patterns in lung ischemia-reperfusion injury (IRI). Toll-like receptors (TLR) are a family of pattern recognition receptors that identify numerous microbial pathogens and form a cornerstone of innate immune defenses. Roles for TLR have been suggested in other organ-based models of IRI. However, the function of TLR in lung IRI is not yet known. We have previously demonstrated that p38 and JNK activation are discretely associated with the alveolar macrophage (AM), while ERK 1/2 activation localizes to non-AM cell types, specifically pulmonary artery endothelial (PAEC) and epithelial cells (type 2 pneumocytes, T2P). This differential pattern of MAPK activation in AM vs. non-AM cells may reflect differential requirements for innate immune receptors such as TLR2 and TLR4 in AM vs. non-AM cells. Using our well developed model of lung IRI in the rat, we developed a method of specific molecular deletion in the AM in vivo using short interference RNA (siRNA). Coupled with in vitro TLR molecular deletion, these methods provide us a unique opportunity to study specific TLR activation requirements in lung IRI.
METHODS: Rats were pre-treated with TLR2 and TLR4 specific siRNA and then subjected to 90 minutes of left lung warm ischemia followed by 15 minutes to 4 hours of reperfusion. Lungs were subsequently explanted and assessed for parameters of lung injury, MAPK activation, and TLR knockdown. Primary AM cultures, PAEC and T2P cultures were pretreated with TLR2 and TLR4 specific siRNA. Following 24 hours of incubation, cells were placed in a hypoxic incubator (0.5% FiO2; 37°c) for 90 minutes, followed by either 15 minutes or 4 hours of reoxygenation. At the termination of the experiments, media was collected for cytokine analysis and cell lysates collected for assessment of MAPK activation patterns and TLR knockdown.
RESULTS: There was a 90% reduction in vascular permeability in TLR4 siRNA treated rats. With TLR4 knockdown there was no appreciable p38 or JNK phosphorylation detected by Western blot analysis in vivo. TLR2 knockdown in vivo did not confer protection from IRI and did not alter p38 or JNK phosphorylation. p38 and JNK phosphorylation persisted in the AM despite TLR2 knockdown. ERK phosphorylation was significantly diminished in TLR2 siRNA treated PAEC and T2P compared to positive controls. TLR2 knockdown did not alter the secretion of the inflammatory cytokine CINC in the AM but decreased CINC secretion in PAEC and T2P by 76% and 78% respectively. Western blotting of total protein confirmed TLR2 and TLR4 knockdown.
CONCLUSIONS: Our results suggest a differential role for TLR2 and TLR4 dependent signaling in response to lung IRI. TLR4 activation in the AM is the centrally important early step in transducing oxidative stress in the lung. This data also suggests a role for a separate innate immune receptor, TLR2, in the proinflammatory response of non-AM cell types to oxidative stress. The development of methods using siRNA provides a powerful tool for determining the precise TLR signaling requirements following lung IRI.
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