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Screening for peptides targeted to IL-7R? for molecular imaging of rheumatoid arthritis synovium.

ABSTRACT: Interleukin-7 receptor alpha (IL-7R?) represents a biomarker with potential applications in rheumatoid arthritis (RA) diagnosis and therapy. We have therefore searched by phage display potential IL-7R? specific peptides with the primary goal being to develop in vivo molecular imaging tools.IL-7R?-targeted peptides were searched within a disulfide-constrained combinatorial phage displayed library of random linear heptapeptides. The apparent dissociation constant (Kd) and half maximal inhibition constant (IC50) were estimated for phage clones and synthesized peptides by ELISA. We used 5-Aza-2'-deoxycytidine (ADC)-stimulated Jurkat cells and human synovial tissue from patients with RA for in vitro characterization of peptides. For molecular imaging studies performed by magnetic resonance imaging (MRI), experimental arthritis was induced in DBA/1 male mice by immunization with an emulsion of complete Freund's adjuvant and type II collagen from chicken sternal cartilage.After several steps of phage display and peptide screening, two IL-7R?-specific heptapeptides (P258 and P725) were selected from the initial library, based on their affinity for the target (extracellular domain of IL-7R?, which contains a fibronectin type III repeat-like sequence). P258 (a linear peptide obtained by removing the Cys-constraint) had the lowest affinity for fibronectin itself and was therefore proposed for molecular imaging. After grafting to ultra-small superparamagnetic particles of iron oxide (USPIO), P258 produced a strong negative contrast on MRI in mice with collagen-induced arthritis (CIA), even at 2 hours post injection. The co-localization of USPIO-P258 with IL-7R?-expressing cells in the synovial tissue from CIA mice and its ability to discriminate the level of IL-7R expression and the disease severity confirmed its efficacy as an in vivo IL-7R? imaging agent. Interestingly, the cyclic peptide (P725), which was less adequate for molecular imaging because of higher affinity for fibronectin, had a strong ability to compete with IL-7 for the IL-7R? binding sites, making it a potential candidate for blocking applications. Accordingly, P725 prevented the signal transducer and activator of transcription 5 (STAT5) activation induced by IL-7 in ADC-stimulated Jurkat cells.The two peptides identified in this work demonstrate that IL-7R? targeting in RA presents potential applications for in vivo molecular imaging and putative blocking purposes.

SUBMITTER: Burtea C

PROVIDER: S-EPMC5059943 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Screening for peptides targeted to IL-7Rα for molecular imaging of rheumatoid arthritis synovium.

Burtea Carmen C Laurent Sophie S Sanli Tuba T Fanfone Deborah D Devalckeneer Aude A Sauvage Sébastien S Beckers Marie-Claire MC Rorive Sandrine S Salmon Isabelle I Vander Elst Luce L Lauwerys Bernard R BR Muller Robert N RN

Arthritis research & therapy 20161012 1

<h4>Background</h4>Interleukin-7 receptor alpha (IL-7Rα) represents a biomarker with potential applications in rheumatoid arthritis (RA) diagnosis and therapy. We have therefore searched by phage display potential IL-7Rα specific peptides with the primary goal being to develop in vivo molecular imaging tools.<h4>Methods</h4>IL-7Rα-targeted peptides were searched within a disulfide-constrained combinatorial phage displayed library of random linear heptapeptides. The apparent dissociation constant ...[more]

PMID: 27729062

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Project description:INTRODUCTION:Angiogenesis is an early event in the pathogenesis of both psoriatic arthritis (PsA) and rheumatoid arthritis (RA); however, there are striking differences in blood vessel morphology and activation between the two arthropathies. The aim of this study was to assess if the PsA and RA joint microenvironments differentially regulate endothelial cell function. METHODS:PsA and RA primary synovial fibroblasts (SFC) were isolated from synovial biopsies, grown to confluence, and supernatants harvested and termed 'conditioned media' (CM). Human umbilical vein endothelial cells (HUVEC) were cultured with PsA SFC or RA SFC-CM (20%). HUVEC tube formation, migration, and PBMC adhesion were assessed by matrigel tube formation, wound repair, and PBMC adhesion assays. HUVEC cell surface expression of ICAM, VCAM, and E-Selectin was assessed by flow cytometry. Transcriptome analysis of genes promoting angiogenesis was performed by real-time PCR. Finally, a MSD multiplex angiogenic assay was performed on PsA SFC and RA SFC supernatants. RESULTS:Macroscopic synovitis and vascularity were similar in PsA and RA patients; however, significant differences in vascular morphological pattern were recorded with tortuous, elongated vessels observed in PsA compared to straight regular branching vessels observed in RA. Transcriptome analysis showed strong upregulation of the pro-angiogenic signature in HUVEC primed with PsA SFC-CM compared to RA SFC-CM and basal control. In parallel, paired PsA SFC-CM significantly induced HUVEC tube formation compared to that of RA SFC-CM. Furthermore, PsA SFC-CM induced HUVEC migration was paralleled by a significant induction in VEGFA, PFKFB3, ICAM-1, and MMP3 mRNA expression. A significant increase in PBMC adhesion and cell surface expression of VCAM-1, ICAM-1, and E-Selectin expression was also demonstrated in PsA SFC-CM-primed HUVEC compared to RA SFC-CM. Finally, VEGF, TSLP, Flt-1, and Tie-2 expression was elevated in PsA SFC-CM compared to RA SFC-CM, with no significant difference in other pro-angiogenic mediators including MIP-3, bFGF, PIGF, and MCP-1. CONCLUSION:PsA SFC and RA SFC secreted factors differentially regulate endothelial cell function, with soluble mediators in the PsA joint microenvironment inducing a more pro-angiogenic phenotype compared to the RA.

Dataset Information

Screening for peptides targeted to IL-7R? for molecular imaging of rheumatoid arthritis synovium.

Publications

Screening for peptides targeted to IL-7Rα for molecular imaging of rheumatoid arthritis synovium.

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