Regulation of CXCL8 function during inflammation

Abstract

Ischaemia-Reperfusion Injury (IRI) is a major contributor to acute organ dysfunction. Chemokines play a key role in leukocyte recruitment to the injured tissue. CXCL8 is a critical inflammatory mediator of neutrophil migration in a range of transplant surgeries. In addition to binding to their G protein-coupled receptor (GPCR) on the leukocyte surface, chemokines also interact with endothelial surface glycosaminoglycan (GAG). Chemokine activity can be regulated by several means, including binding to GPCRs, GAGs, and through post-translational modifications (PTMs). GAGs bind and immobilise chemokines at high concentrations at the site of injury. This directs the migration and activation of leukocytes, contributing to local inflammation. We aimed to interfere with this binding in order to modulate neutrophil recruitment through the use of chemokine peptides. A truncated version of wild type CXCL8 corresponding to the GAG-binding region of CXCL8, located towards the C-terminal α-helix, the E70K peptide and a scrambled peptide were chemically synthesised (90% purity). Their GAG binding was shown by Surface Plasmon Resonance. Flow-based neutrophil adhesion was significantly decreased by WT peptide, E70K peptide or scrambled peptide (p<0.01), with no significant difference among them, suggesting a competitive role of peptides at displacing CXCL8 from GAG. In vitro neutrophil transendothelial chemotaxis mediated by CXCL8 gradient was decreased by E70K peptide (p<0.001) as opposed to WT or scrambled peptides. Thus, E70K peptide higher positive charge might contribute to higher specificity to bind polyanionic GAG. Post-translational nitration appears to render CXCL8 non-functional, reducing migration completely (p<0.001). The presence of Reactive Nitrogen Species and CXCL8 in kidney biopsies was assessed by immunofluorescence. There was an association between grade of injury and nitration. CXCL8 expression did not significantly change. Our study has generated a better understanding of CXCL8 function, particularly the process involved in targeted competitive modulation and the role of chemokine nitration as a negative regulator. This could offer therapeutic opportunities to protect the organ from neutrophil-derived damage during IRI.