Designing CXCL8-based decoy proteins with strong anti-inflammatory activity in vivo

Abstract

IL (interleukin)-8 [CXCL8 (CXC chemokine ligand 8)] exerts its role in inflammation by triggering neutrophils via its specific GPCRs (G-protein-coupled receptors), CXCR1 (CXC chemokine receptor 1) and CXCR2, for which additional binding to endothelial HS-GAGs (heparan sulphate-glycosaminoglycans) is required. We present here a novel approach for blocking the CXCL8-related inflammatory cascade by generating dominant-negative CXCL8 mutants with improved GAG-binding affinity and knocked-out CXCR1/CXCR2 activity. These non-signalling CXCL8 decoy proteins are able to displace WT (wild-type) CXCL8 and to prevent CXCR1/CXCR2 signalling thereby interfering with the inflammatory response. We have designed 14 CXCL8 mutants that we subdivided into three classes according to number and site of mutations. The decoys were characterized by IFTs (isothermal fluorescence titrations) and SPR (surface plasmon resonance) to determine GAG affinity. Protein stability and structural changes were evaluated by far-UV CD spectroscopy and knocked-out GPCR response was shown by Boyden chamber and Ca2+ release assays. From these experiments, CXCL8(Δ6F17KF21KE70KN71K) emerged with the most promising in vitro characteristics. This mutant was therefore further investigated in a murine model of mBSA (methylated BSA)-induced arthritis in mice where it showed strong anti-inflammatory activity. Based on these results, we propose that dominant-negative CXCL8 decoy proteins are a promising class of novel biopharmaceuticals with high therapeutic potential in inflammatory diseases.

The effect of the decoy molecule PA401 on CXCL8 levels in bronchoalveolar lavage fluid of patients with cystic fibrosis

“…A murine model of mBSA (methylated BSA) -induced arthritis has been utilised to evaluate the systemic anti-inflammatory potential of PA401. The decoy was administered subcutaneously resulting in a decrease in neutrophil infiltration to the knee cavity as measured in knee lavage fluid (Falsone et al, 2013). The results obtained from animal studies thus far have been achieved by delivering the decoy intravenously.…”

“…PA401 (CXCL8 Δ6 F17K F21K E70K N71K) was generated by ProtAffin Biotechnologie AG, Graz, Austria, according to published protocols (Falsone et al, 2013).…”

Anti‐inflammatory effects of the GAG‐binding CXCL9(74‐103) peptide in dinitrofluorobenzene‐induced contact hypersensitivity in mice

“…41 Jose, CA, USA). On the day of the assay, endothelial monolayers were stimulated with 50 ng/mL human TNF-α or mouse TNF-α (Peprotech) for 4 hours at 37°C.…”

99mTc-CXCL8 SPECT to Monitor Disease Activity in Inflammatory Bowel Disease

“…Recently, selective CXCR1 and CXCR2 antagonists have been tested in the clinical setting (32,33), and these targeted drugs could be dosed, based on the results of functional imaging studies. CXCL8-based decoy proteins demonstrated strong antiinflammatory activity in vivo in preclinical models (34). Given the results in this study and the translational potential of imaging immune cell recruitment with 99m Tc-CXCL8 in inflammatory bowel conditions, further prospective studies to validate the tracer specificity and correlation with other noninvasive biomarkers are warranted.…”