Introduction: Exosomes play pivotal roles in intercellular communication, facilitating delivery of proteins and nucleic acids to proximal and distal cells, and are elevated in the circulation following liver ischemia-reperfusion injury (IRI). Dendritic cells (DC) are key regulators of innate and adaptive immunity. They have been shown previously to play key roles in regulation of experimental liver IRI and liver transplant outcome. Here we examined for the first time the influence of exosomes purified from IRI and control livers on DC function.
Methods: Normal C57BL/6 (B6) male WT mice underwent 70% hepatic warm ischemia for one hour, followed by six hours reperfusion. Liver exosomes (<200 nm) were isolated, purified and quantified according to the guidelines of the International Society of Extracellular Vesicles. Western blotting was used to determine exosome purity. Conventional myeloid DC (CD11c+CD11b+NK1.1-) were generated from either normal B6 bone marrow (BMDC) or isolated from normal liver tissue following in situ liver perfusion and collagenase digestion. The DC were pulsed for 24 hours with liver-derived exosomes that were visualized/tracked by confocal microscopy. Subsequently, the DC were analyzed phenotypically by flow cytometry and characterized functionally for allogeneic T cell stimulatory activity in MLR and by quantification of secreted cytokines.
Results: Liver exosomes were increased significantly following IRI. Their purity was verified by expression of the exosome markers CD63 and CD81, and by absence of the endoplasmic reticulum marker glucose-regulated protein (GRP)78.  Exosomes from ischemic livers (but not those from normal control livers) enhanced the activation and maturation of BMDC, evidenced by increased MHC II and co-stimulatory molecule (CD80/86) expression. This correlated with augmented ability to induce allogeneic CD4 and CD8 T cell proliferative responses in MLR, as well as elevated secretion of proinflammatory cytokines (IFNγ, IL-17A, and TNFα). By contrast, normal liver-resident DCs were refractory to liver IRI-exosome stimulation and displayed comparative phenotypic and functional resistance to maturation and acquisition of T cell stimulatory activity.
Conclusion: These novel findings reveal a differential capacity of liver interstitial exosomes released following IRI to affect the phenotype and stimulatory function of bone marrow-derived versus liver-resident myeloid DCs. The refractory response of liver-resident DC may contribute to modulation of tissue injury and attenuation of alloimmune reactivity following liver transplantation.