Mitochondrial Damage-Associated Molecular Patterns (MTDs) Released from Hepatic Ischemia Reperfusion Induce Inflammatory Response

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BackgroundDuring liver transplantation ischemia and reperfusion injury (IRI), an immune driven inflammatory response provoked by cellular oxygen deprivation, is unavoidable. The inflammatory responses, resulting from acute oxidative stress and consequent hepatocellular death during the early reperfusion phase, cause the release of damage-associated molecular patterns. Recent studies show that the release of MTDs is directly linked with functionally important immune consequences and injury. We investigated the roles of MTDs in liver IRI.MethodsRat and human hepatocytes were used in the in vitro models, mice were used in an in vivo model of hepatic IR. Liver perfusate samples were obtained from porcine and human normothermic ex vivo liver perfusion (NMP) transplants from our collaborators and correlated to outcomes. MTDs were extracted and quantified by qPCR and PicoGreen assay. Liver biopsies were assayed by transmission electron microscopy (TEM) to assess for damaged mitochondria. MitoTracker was used to test the mitochondrial homeostasis by flow cytometry. The hepatocytes or macrophages were co-cultured with different concentrations of MTDs, and cell viability was demonstrated by apoptosis detection kit; the inflammatory cytokines (TNF-α, IL-6, and IL-1β) were tested by ELISA.ResultsThe MTDs levels were significantly higher in IRI in vitro, and marked mitochondria damage was detected by TEM and flow cytometry. Co-culture of MTDs with hepatocytes significantly increased the cell death in a dose-dependent manner. Co-culture MTDs with macrophages significantly increased proinflammatory cytokine release via TLRs/MyD88 NF-κB pathway. The release of MTDs detected in vivo was associated with inflammatory cytokine secretion and more severe liver injury compared to the control. MTDs acted as an alarmin after hepatic IRI, which might contribute to the initiation of systemic inflammatory response. MTDs levels from pig DCD perfusate were higher than DBD perfusate samples in the pig NMP model, and higher level of MTDs were associated with worse clinical outcome in both the pig and human liver transplants following NMP.ConclusionOur results indicate that IRI can elicit a significant increase of MTDs both in vitro and in vivo, and that MTDs in ex vivo perfusates can be used as potential biomarkers of hepatic IRI and may predict outcomes after liver transplantation.

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