Down-regulation of inducible co-stimulator (ICOS) by intravitreal injection of small interfering RNA (siRNA) plasmid suppresses ongoing experimental autoimmune uveoretinitis in rats |
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Authors: | Yongsheng Hou Lin Xing Shaoying Fu Xiaoning Zhang Jingjing Liu Hongling Liu Bingjie Lv Hao Cui |
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Institution: | (1) Department of Ophthalmology, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001, People’s Republic of China;(2) Heilongjiang Provincial Institute of Drug Control, Harbin, People’s Republic of China |
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Abstract: | Background RNA interference (RNAi) is now being exploited as a powerful tool for gene knockdown. Recently, we had shown that inducible
co-stimulator (ICOS) was up-regulated in experimental autoimmune uveoretinitis (EAU). The aim of this study was to investigate
whether intravitreal injection of small interfering RNA (siRNA) plasmid, targeting ICOS, suppresses the ongoing experimental
autoimmune uveoretinitis (EAU) in rats.
Methods Oligonucleotide targeting ICOS was cloned into linearized pRNAT-U6.1/Neo eukaryotic expression vector to construct the recombinant
plasmid (pRNAT-U6.1/Neo-ICOS). After transfecting activated rat T cells with the recombinant plasmid, ICOS mRNA and protein
expression levels were determined by real-time RT-PCR and Western blot analysis respectively. Rats were immunized with IRBP
R16 peptide emulsified in complete Freund’s adjuvant (CFA) and given an intravitreal injection of pRNAT-U6.1/Neo-ICOS on day
6 after immunization. After 13days of immunization, the ICOS protein expression and CD4 + ICOS + T cells were identified in retinae through Western blot analysis and flow cytometry respectively. Intraocular inflammation
was assessed by the scores of the clinical and histological appearances. Delayed-type hypersensitivity (DTH) and lymphocyte
proliferation were detected to evaluate the systemic effect of intravitreal injection of pRNAT-U6.1/Neo-ICOS.
Result The recombinant plasmid (pRNAT-U6.1/Neo-ICOS) for the ICOS siRNA was successfully constructed. In vitro studies using the
recombinant plasmid has showed the down-regulation of ICOS gene expression both at the mRNA and protein levels. Clinical and
pathological scores showed that ocular inflammation of pRNAT-U6.1/Neo-ICOS-treated eyes was markedly less than that of vehicle-treated
eyes. The expression of ICOS protein and the amount of CD4 + ICOS + T cells in retinae significantly decreased by intravitreal injection of the recombinant plasmid, whereas delayed-type hypersensitivity
response and lymphocyte proliferation were not impaired in rats treated with the recombinant plasmid.
Conclusion Intravitreal injection of siRNA plasmid targeting ICOS effectively down-regulated the expression of ICOS, and was highly effective
in suppressing the ongoing process of EAU without any side-effects on systemic cellular immunity.
Yongsheng Hou and Lin Xing contributed equally to this study |
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Keywords: | Inducible co-stimulator (ICOS) Experimental autoimmune uveoretinitis (EAU) RNA interference (RNAi) |
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