Down-regulation of inducible co-stimulator (ICOS) by intravitreal injection of small interfering RNA (siRNA) plasmid suppresses ongoing experimental autoimmune uveoretinitis in rats.
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.


RESULTS
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.


CONCLUSIONS
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.
Hou
Yongsheng
Y
Department of Ophthalmology, The First Affiliated Hospital of Harbin Medical University, Harbin, People's Republic of China.
Xing
Lin
L
Fu
Shaoying
S
Zhang
Xiaoning
X
Liu
Jingjing
J
Liu
Hongling
H
Lv
Bingjie
B
Cui
Hao
H
D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics* / Q000378:metabolism; D001327:Autoimmune Diseases / Q000276:immunology / Q000473:pathology / Q000517:prevention & control*; D015153:Blotting, Western; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018929:Cell Culture Techniques; D004195:Disease Models, Animal; D015536:Down-Regulation / Q000187:drug effects*; D005260:Female; D005434:Flow Cytometry; D020868:Gene Silencing; D006968:Hypersensitivity, Delayed / Q000276:immunology; D060889:Inducible T-Cell Co-Stimulator Protein; D007267:Injections; D008213:Lymphocyte Activation; D010446:Peptide Fragments; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics; D051381:Rats; D011917:Rats, Inbred Lew; D012173:Retinitis / Q000276:immunology / Q000473:pathology / Q000517:prevention & control*; D012177:Retinol-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014605:Uveitis / Q000276:immunology / Q000473:pathology / Q000517:prevention & control*; D014822:Vitreous Body
D000945:Antigens, Differentiation, T-Lymphocyte; C556695:Icos protein, rat; D060889:Inducible T-Cell Co-Stimulator Protein; D010446:Peptide Fragments; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012177:Retinol-Binding Proteins; C084432:immunodominant R16 protein, rat