Chemokine-gebaseerde glycosaminoglycaan bindende peptiden: een nieuwe strategie voor de inhibitie van inflammatie

Chemokines or chemotactic cytokines are proteins (8-14 kDa) which are known to play an important role in the migration of leukocytes and can be classified, based on their function, into homeostatic and inflammatory chemokines. Inflammatory chemokines are locally secreted by tissue cells and resident leukocytes upon infection or tissue damage, thereby creating a gradient along which leukocytes can migrate from the blood vessels to the site of inflammation. To prevent washout by the blood flow and to create such a directional gradient, it is necessary that chemokines are presented on the endothelium and immobilized in the tissue at the site of inflammation through binding to glycosaminoglycans (GAGs). This interaction ensures high local concentrations of the produced chemokines, prevents their diffusion and inhibits proteolytic degradation. Subsequently, chemokines interact with their G protein-coupled receptor(s) (GPCRs), specifically expressed by circulating leukocyte‑subtypes, which results in adhesion of leukocytes and extravasation through the endothelium. Once leukocytes enter the tissue, they can migrate to the site of inflammation through a gradient of GAG-bound chemokines. The binding of chemokines to GAGs has been proven to be indispensable for chemokine activity in vivo. Chemokines and their GPCRs have served as potential targets for the development of new anti-inflammatory drugs over the past 20 years. However, this was not very successful since only a few chemokine receptor antagonist are currently used as therapeutics. Therefore, a new strategy to target the chemokine system, stressing chemokine-GAG interactions, was being explored. At first, we identified several COOH-terminally truncated CXCL9 isoforms in conditioned medium of fibroblasts or peripheral blood mononuclear cells (PBMCs) stimulated with inflammatory agents. Interestingly, all these CXCL9 isoforms lost their characteristic cationic COOH-terminal region. Since chemokine-GAG interactions are mainly mediated by the interaction of basic amino acids in the chemokine and negatively charged sulfate groups in the GAG, the high number of positive charges in the COOH-terminal part of CXCL9 suggested a GAG binding function. To evaluate this, several COOH-terminal CXCL9-derived peptides, such as CXCL9(74‑103), CXCL9(74-93) and CXCL9(86‑103), were chemically synthesized. Both CXCL9(74-103) and CXCL9(74-93) bound with high but different affinity to GAGs. In addition, these peptides were able to compete with active chemokines for GAG binding in an ELISA-like heparin binding assay and were able to inhibit the in vivo recruitment of neutrophils towards CXCL8 injected in the peritoneum or the knee cavity. However, reducing the length of the CXCL9 peptide gradually decreased the capacity to compete with CXCL8 for GAG binding. CXCL9(86-103) failed to inhibit CXCL8‑induced neutrophil recruitment in vivo underlining the importance of the two typical heparin binding motifs, present in CXCL9(74-103). Although CXCL9(74‑93) contains the same GAG binding motifs as CXCL9(74-103), this peptide was unable to inhibit MSU crystal-induced neutrophil migration to the knee cavity whereas the CXCL9(74-103) peptide could. It was also shown that the CXCL9(74-103) peptide coats the endothelial cell surface in vivo thereby inhibiting CXCL8-induced neutrophil adhesion in the murine cremaster muscle model. In another neutrophil-dependent inflammation model, a model of dinitrofluorobenzene (DNFB)‑induced contact hypersensitivity, CXCL9(74-103) decreased neutrophil infiltration and ear swelling. In addition to its anti-inflammatory role, we showed that such positively charged chemokine‑derived peptides have antiviral activity against several viruses, such as DENV serotype 2, clinical and laboratory strains of HSV-1 and RSV. This is explained by the importance of GAG binding for virus infection. Indeed, the CXCL9(74-103) peptide competed with an envelope protein of DENV for binding to heparin, thereby blocking the entry of the virus in the host cell. In conclusion, in this doctoral thesis we identified a novel approach to target the chemokine system to reduce inflammation. The chemokine-derived GAG binding peptides block the binding of active chemokines to GAGs, thereby inhibiting the chemokine activity in vivo. This reduced leukocyte migration eventually leads to a reduction in leukocyte-dependent inflammation. Therefore, these chemokine-derived peptides could serve as lead molecules for the generation of new anti-inflammatory therapeutics.nrpages: 174status: publishe