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.
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Authors | Yongsheng Hou, Lin Xing, Shaoying Fu, Xiaoning Zhang, Jingjing Liu, Hongling Liu, Bingjie Lv, Hao Cui |
Journal | Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie
(Graefes Arch Clin Exp Ophthalmol)
Vol. 247
Issue 6
Pg. 755-65
(Jun 2009)
ISSN: 1435-702X [Electronic] Germany |
PMID | 19125271
(Publication Type: Journal Article)
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Chemical References |
- Antigens, Differentiation, T-Lymphocyte
- Icos protein, rat
- Inducible T-Cell Co-Stimulator Protein
- Peptide Fragments
- RNA, Messenger
- RNA, Small Interfering
- Retinol-Binding Proteins
- immunodominant R16 protein, rat
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Topics |
- Animals
- Antigens, Differentiation, T-Lymphocyte
(genetics, metabolism)
- Autoimmune Diseases
(immunology, pathology, prevention & control)
- Blotting, Western
- CD4-Positive T-Lymphocytes
(immunology)
- Cell Culture Techniques
- Disease Models, Animal
- Down-Regulation
(drug effects)
- Female
- Flow Cytometry
- Gene Silencing
- Hypersensitivity, Delayed
(immunology)
- Inducible T-Cell Co-Stimulator Protein
- Injections
- Lymphocyte Activation
- Peptide Fragments
- Plasmids
- RNA Interference
- RNA, Messenger
(genetics, metabolism)
- RNA, Small Interfering
(administration & dosage, genetics)
- Rats
- Rats, Inbred Lew
- Retinitis
(immunology, pathology, prevention & control)
- Retinol-Binding Proteins
- Reverse Transcriptase Polymerase Chain Reaction
- Transfection
- Uveitis
(immunology, pathology, prevention & control)
- Vitreous Body
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