Inhibition of Experimental Autoimmune Uveoretinitis (EAU) in Rat by Electrotransfer of Ciliary Muscle by hTNF-alpha Soluble Receptor Gene

Abstract

Purpose: : Systemic administration of p55 TNF receptor fusion protein (hTNFr-Ig) is effective for treating patients with posterior uveitis but is potentially associated to systemic side effects. We showed that plasmid electrotransfer (ET) of ciliary muscle with hTNF-alpha soluble receptor gene allowed a sustained protein secretion restricted to the eye (Bloquel, FASEB J., 2006). The aim of this study was to evaluate the effect of ET of hTNFr-Ig on EAU.

Methods: : EAU was induced in Lewis rats by subcutaneous injection of S-Ag in complete Freund’s adjuvant and treatment of both eyes was started 4 days before immunization. Intramuscular injection was performed in the ciliary muscle followed by ET. An iridium/platinum needle cathode was placed at the injection site and the anode was placed on the sclera using 200V/cm field. Control eyes received ET of the backbone (pVAX2) (n=12), and treated eyes received injection of pVAX2-hTNFr-Ig (n=18) or ET of pVAX2-hTNFr-Ig (30µg) (n=18). Clinical disease severity was scored and at day 21 after immunization, eyes were enucleated for histology and immunohistochemical analysis by ED1/iNOS labeling. Aqueous humor / vitreous were also collected for cytokine and chemokine analysis by multiplex ELISA.

Results: : The local hTNFr-Ig secretion into the eyes of rats treated with ET of pVAX2-hTNFr-Ig delayed the onset of EAU (day 17±1.1, p=0.03) compared to the control group treated with ET of pVAX-2 (day 13±0.3) and reduced (p=0.02) histological retinal damages (scores: ET pVAX2-TNFr-Ig:1.6±0.4; ET pVAX-2: 3.4±0.6). In contrast, the injection of pVAX2-hTNFr-Ig had no effect on EAU. Compared to controls, low level (p<0.05) of CCL2, CCL5 and IL-17 were found in ocular media of eyes treated by ET of hTNFr-Ig together with higher level (p<0.05) of Th2 cytokine (IL10, IL-13 and IL-4) and IFN-gamma. In ocular sections of treated rats, low number of ED1+ macrophages showing reduced iNOS expression was found implying change of their phenotype.

Conclusions: : hTNFr-Ig plasmid ET efficiently reduced EAU severity through reduction of local chemokine expression thus reducing inflammatory cell infiltration. An increase of regulatory cytokines in the treated eyes with reduced activity of macrophages could explain the beneficial effect of ET of pVAX2-hTNFr-Ig on EAU.

Keywords: gene transfer/gene therapy • immunomodulation/immunoregulation • cytokines/chemokines