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LPS Preconditioning Confers Ischemic Tolerance in the Lung Primarily Through a MyD88-Independent Pathway
Elizabeth FitzSullivan, John C Keech, Heather E Merry, Patrick S Wolf, *Michael S Mulligan
U. of Washington, Seattle, WA
BACKGROUND: Lung transplantation is an effective treatment for select patients with end-stage pulmonary disease, yet availability of donor organs remains a barrier to transplantation. While overt infection is considered a contraindication, the implications of an isolated positive gram stain remain unclear. LPS, the best known ligand for TLR-4, has been shown to induce acute lung injury in high doses, while paradoxically, low doses may modulate subsequent lung ischemia reperfusion injury (LIRI). TLR-4 classically initiates signaling through recruitment of the adaptor protein MyD88, but can also initiate a MyD88 independent signaling pathway, reducing proinflammatory signaling and leading to type 1 interferon responses and IL-10 production, which are protective in LIRI. We hypothesize that LPS preconditioning alters the TLR-4 signaling response to oxidative stress by signaling through a MyD88-independent pathway, thereby conferring ischemic tolerance.
METHODS: Rats were pretreated with intravenous MyD88 or TLR-4 siRNA in a lipid vector 48 hours prior to low dose intratracheal LPS installation and prior to left lung ischemia and reperfusion (IR). Following reperfusion, lungs were assessed for vascular permeability, cytokine content, MAPK activation and target protein knockdown.
RESULTS: LPS pretreatment followed by the IR protocol led to a 74% reduction in vascular permeability (p<0.05), a greater than 90% reduction in CINC and TNF-α secretion (p<0.001), and an 80% reduction in both JNK and p38 phosphorylation (p<0.05), compared to positive controls. LPS pretreatment without IR yielded results equivalent to negative controls. MyD88 knockdown did not diminish the protective effect of LPS pretreatment, whereas TLR-4 knockdown did eliminate the protective effect. Western blotting confirmed effective knockdown of MyD88 and TLR-4.
CONCLUSIONS: Low dose LPS pretreatment significantly reduced endothelial dysfunction and inflammatory mediator production following subsequent exposure to IR. This work is the first to describe a functionally relevant, protective effect of LPS preactivation on LIRI through a TLR-4 mediated MyD88-independent pathway. Understanding the protective effect of LPS preconditioning has the potential to expand and clarify donor inclusion criteria, therefore improving the clinical outcome of patients with end-stage pulmonary disease. 
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