Background:Although Galecin 9 (Gal-9) negatively regulates Th1-type alloimmunity by binding to T-cell Immunoglobulin Mucin-3 (TIM-3) on activated Th1 cells, its role in liver ischemia-reperfusion injury (IRI) remains elusive. Methods/Results: First, we assessed the kinetics of Gal-9 expression in a mouse model of hepatic warm ischemia (90min). Unlike in sham controls, the expression of Gal-9 mRNA increased progressively during the reperfusion, peaking at 6h and decreasing thereafter (12-24h). This correlated with the kinetics of hepatocellular damage (sALT), and local innate immune activation pattern. Immunofluorescence staining showed that unlike in sham-livers, dense Gal-9 expression was detectable in IR-stressed livers at 6h, primarily in hepatocytes, and as a secreted protein in sinusoidal liver vessels. Second, to confirm in-vivo findings, we probed for Gal-9 expression in mouse primary hepatocyte cultures. Unlike untreated hepatocytes, those that were subjected to H₂O₂ stress, strongly expressed Gal-9. Third, we employed WT and TIM-3Tg (B6) mice in a model of extended (20h) hepatic cold storage (4C) and orthotopic liver transplantation (OLT). Groups of WT and TIM-3Tg mice were pre-conditioned with a single dose of human stable-form rGal-9 (100ug/mouse i.v. at -1h). Administration of rGal-9 attenuated IRI in OLTs, evidenced by sALT levels (764.50±145.98 IU/L vs. 7802.33±1515.33 IU/L in untreated WT; p<0.01) by 6h of reperfusion. Adjunctive rGal-9 further enhanced cytoprotection in IR-stressed TIM-3Tg livers (sALT: 335.27±107.77 IU/L vs. 1368.33±217.82 IU/L in untreated TIM-3Tg; p<0.01). Gal-9 therapy also diminished IR-mediated WT and TIM-3Tg OLT damage, as shown by histology. Interestingly, pretreatment with rGal-9 blunted innate immune activation by decreasing mRNA levels coding for TLR4, TNF-α, IL-1β and IL-6 (p<0.05) in IR-stressed WT and TIM-3Tg OLTs. Finally, we analyzed Gal-9 function in-vitro. Indeed, addition of rGal-9 diminished ConA-induced T cell activation in WT and TIM-3Tg cell cultures, evidenced by decreased IFN-γ levels (ELISA) and increased CD4+ T cell apoptosis (caspase-3/7 expression by FACS). Conclusion: Harnessing Gal-9 – TIM-3 signaling at the hepatocyte – T cell interface facilitates homeostasis in IR-stressed OLTs. Enhanced expression of hepatocyte Gal-9 potentiates resistance against liver IRI. These results offer the rationale for refined novel strategies to combat IRI in organ transplant recipients.

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