Xenotransplantation is a rapidly growing scientific field whose goal is to solve the shortage of organs available for transplantation by using pig organs in place of human organs. However, acute neutrophil sequestration is consistently associated with xenograft lung injury, despite genetic modifications of the donor organ. Sialic acid levels regulate cellular adhesive mechanisms in inflammatory or tumor environments. Here we asked if sialic acid removal promotes xenogeneic neutrophil adhesion and examined the role of galectin-3.
The adhesion of human neutrophils to genetically modified (GTKO.hCD46) pig aortic endothelial cells (pAECs) was measured in static and microfluidic flow chamber conditions (Bioflux, 1dyne/cm2) in presence of various treatments.
Desialation of pAECs or calceinAM labelled neutrophils with Clostridium perfringens neuraminidase (NA, 25mU/mL) increased neutrophil adhesion (36% vs. 22%, p<0.0001). In contrast, pan-neuraminidase inhibition decreased neutrophil adhesion: treatment of pAECs with 2-deoxy-NANA (DANA, 1mM) almost completely prevented adhesion, whereas treatment of neutrophils with DANA reduced adhesion by ~60%. N-acetyllactosamine, which inhibits galectin binding, decreased neutrophil adhesion to NA-pretreated pAECs (14% vs. 36%, p<0.0001). Pig galectin-3 mRNA levels were increased after lung perfusions. Incubating both pAECs and neutrophils with anti-human galectin-3 antibody completely abrogated NA-dependent adhesion to human tumor necrosis factor-treated (25ng/mL) pAECs (NA/gal-3: 22% vs. NA: 66%, no NA: 30%, p<0.0001).
Modulation of cellular sialation regulates human neutrophil adhesion to pAECs, such that inhibiting sialidase activity may prevent neutrophil-mediated inflammation and tissue injury in xenogeneic models. This work further identifies galectin-3 as a candidate for therapeutic interventions to limit neutrophil adhesion in xenotransplantation models.
Benjamin Cerel– MGH and Harvard Medical School
Beth French– University of Maryland Baltimore
Christopher Laird– University of Maryland Baltimore
Xiangfei Cheng– University of Maryland Baltimore
Lars Burdorf– MGH and Harvard Medical School
Richard Pierson– MGH and Harvard Medical School
Agnes Azimzadeh– MGH and Harvard Medical School