RESUMO

Ischemia-reperfusion injury (IRI) drives graft rejection and is the main cause of mortality after liver transplantation. During IRI, an intense inflammatory response marked by chemokine production and neutrophil recruitment occurs. However, few strategies are available to restrain this excessive response. Here, we aimed to interfere with chemokine function during IRI in order to disrupt neutrophil recruitment to the injured liver. For this, we utilized a potent glycosaminoglycan (GAG)-binding peptide containing the 30 C-terminal amino acids of CXCL9 (MIG30) that is able to inhibit the binding of chemokines to GAGs in vitro. We observed that mice subjected to IRI and treated with MIG30 presented significantly lower liver injury and dysfunction as compared to vehicle-treated mice. Moreover, the levels of chemokines CXCL1, CXCL2 and CXCL6 and of proinflammatory cytokines TNF-α and IL-6 were significantly reduced in MIG30-treated mice. These events were associated with a marked inhibition of neutrophil recruitment to the liver during IRI. Lastly, we observed that MIG30 is unable to affect leukocytes directly nor to alter the stimulation by either CXCL8 or lipopolysaccharide (LPS), suggesting that its protective properties derive from its ability to inhibit chemokine activity in vivo. We conclude that MIG30 holds promise as a strategy to treat liver IRI and inflammation.

Assuntos

Quimiocinas , Traumatismo por Reperfusão , Animais , Quimiocinas/metabolismo , Isquemia/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/metabolismo , Peptídeos/farmacologia , Reperfusão/efeitos adversos , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/prevenção & controle

RESUMO

BACKGROUND: Ischemia-reperfusion (IR) is a major contributor to graft rejection after liver transplantation. During IR injury, an intense inflammatory process occurs in the liver. Neutrophils are considered central players in the events that lead to liver injury. CXC chemokines mediate hepatic inflammation following reperfusion. However, few studies have demonstrated in real-time the behavior of recruited neutrophils. We used confocal intravital microscopy (IVM) to image neutrophil migration in the liver and to analyze in real-time parameters of neutrophil recruitment in the inflamed tissue in animals treated or not with reparixin, an allosteric antagonist of CXCR1/2 receptors. MATERIALS AND METHODS: WT and LysM-eGFP mice treated with reparixin or saline were subjected to 60 min of ischemia followed by different times of reperfusion. Mice received Sytox orange intravenously to show necrotic DNA in IVM. The effect of reparixin on parameters of local and systemic reperfusion-induced injury was also investigated. RESULTS: IR induced liver injury and inflammation, as evidenced by high levels of alanine aminotransferase and myeloperoxidase activity, chemokine and cytokine production, and histological outcome. Treatment with reparixin significantly decreased neutrophil influx. Moreover, reparixin effectively suppressed the increase in serum concentrations of TNF-α, IL-6, and CCL3, and the reperfusion-associated tissue damage. The number of neutrophils in the liver increased between 6 and 24 h of reperfusion, whereas the distance traveled, velocity, neutrophil size and shape, and cluster formation reached a maximum 6 h after reperfusion and then decreased gradually. In vivo imaging revealed that reparixin significantly decreased neutrophil infiltration and movement and displacement of recruited cells. Moreover, neutrophils had a smaller size and less elongated shape in treated mice. CONCLUSION: Imaging of the liver by confocal IVM was successfully implemented to describe neutrophil behavior in vivo during liver injury by IR. Treatment with reparixin decreased not only the recruitment of neutrophils in tissues but also their activation state and capacity to migrate within the liver. CXCR1/2 antagonists may be a promising therapy for patients undergoing liver transplantation.