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 smal...
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| creator | Hou, Yongsheng Xing, Lin Fu, Shaoying Zhang, Xiaoning Liu, Jingjing Liu, Hongling Lv, Bingjie Cui, Hao |
| description | 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. |
| doi_str_mv | 10.1007/s00417-008-1023-0 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_20587955</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>20587955</sourcerecordid><originalsourceid>FETCH-LOGICAL-c466t-863c0d7403e743ce3ede1ac2c3fdfdb5d38932f82e6d5fcc3c9c74e8e9973c973</originalsourceid><addsrcrecordid>eNp1kctq3DAUhk1paaZpH6CbIroIyUKtLvbIXobpLRAa6AWyExr5eNBgS66OlDYP1verzEwJFLo6unz_r3P0V9VLzt5wxtRbZKzmijLWUs6EpOxRteK1bKhi4vZxtWJKcNpKcXtSPUPcs4LLhj-tTnjHRSMUX1W_34WfnkbY5dEkFzwJA3G-z9ZtRyA2UExuWu5CJOdXm5uvF2R7X4gUzZ1LEcxYNnuwf7U4mXE5ShAHiM7vyJfPl-QcXSkXZB4NTq4nmOc5AiIgCX4XFgx-zYWfwKdiaXIKbpqyB5LvIERIzrvksBiTaBI-r54MZkR4cayn1fcP779tPtHrm49Xm8trauv1OtF2LS3rVc0kqFpakNADN1ZYOfRDv2162XZSDK2Add8M1krbWVVDC12nylrJ0-rs4DvH8CMDJj05tDCOxkPIqAVrWtU1TQFf_wPuQ46-9KaFZAXp2rpA_ADZGBAjDHouE5t4rznTS6D6EKgugeolUM2K5tXROG8n6B8UxwQLIA4Azst3Q3x4-f-ufwCqYrAA</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>230795984</pqid></control><display><type>article</type><title>Down-regulation of inducible co-stimulator (ICOS) by intravitreal injection of small interfering RNA (siRNA) plasmid suppresses ongoing experimental autoimmune uveoretinitis in rats</title><source>MEDLINE</source><source>SpringerLink Journals - AutoHoldings</source><creator>Hou, Yongsheng ; Xing, Lin ; Fu, Shaoying ; Zhang, Xiaoning ; Liu, Jingjing ; Liu, Hongling ; Lv, Bingjie ; Cui, Hao</creator><creatorcontrib>Hou, Yongsheng ; Xing, Lin ; Fu, Shaoying ; Zhang, Xiaoning ; Liu, Jingjing ; Liu, Hongling ; Lv, Bingjie ; Cui, Hao</creatorcontrib><description>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.</description><identifier>ISSN: 0721-832X</identifier><identifier>EISSN: 1435-702X</identifier><identifier>DOI: 10.1007/s00417-008-1023-0</identifier><identifier>PMID: 19125271</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer-Verlag</publisher><subject>Animals ; Antigens, Differentiation, T-Lymphocyte - genetics ; Antigens, Differentiation, T-Lymphocyte - metabolism ; Autoimmune Diseases - immunology ; Autoimmune Diseases - pathology ; Autoimmune Diseases - prevention & control ; Basic Science ; 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 ; Medicine ; Medicine & Public Health ; Ophthalmology ; Peptide Fragments ; Plasmids ; Rats ; Rats, Inbred Lew ; Retinitis - immunology ; Retinitis - pathology ; Retinitis - prevention & control ; Retinol-Binding Proteins ; Reverse Transcriptase Polymerase Chain Reaction ; RNA Interference ; RNA, Messenger - genetics ; RNA, Messenger - metabolism ; RNA, Small Interfering - administration & dosage ; RNA, Small Interfering - genetics ; Transfection ; Uveitis - immunology ; Uveitis - pathology ; Uveitis - prevention & control ; Vitreous Body</subject><ispartof>Graefe's archive for clinical and experimental ophthalmology, 2009-06, Vol.247 (6), p.755-765</ispartof><rights>Springer-Verlag 2008</rights><rights>Springer-Verlag 2009</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c466t-863c0d7403e743ce3ede1ac2c3fdfdb5d38932f82e6d5fcc3c9c74e8e9973c973</citedby><cites>FETCH-LOGICAL-c466t-863c0d7403e743ce3ede1ac2c3fdfdb5d38932f82e6d5fcc3c9c74e8e9973c973</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00417-008-1023-0$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00417-008-1023-0$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19125271$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hou, Yongsheng</creatorcontrib><creatorcontrib>Xing, Lin</creatorcontrib><creatorcontrib>Fu, Shaoying</creatorcontrib><creatorcontrib>Zhang, Xiaoning</creatorcontrib><creatorcontrib>Liu, Jingjing</creatorcontrib><creatorcontrib>Liu, Hongling</creatorcontrib><creatorcontrib>Lv, Bingjie</creatorcontrib><creatorcontrib>Cui, Hao</creatorcontrib><title>Down-regulation of inducible co-stimulator (ICOS) by intravitreal injection of small interfering RNA (siRNA) plasmid suppresses ongoing experimental autoimmune uveoretinitis in rats</title><title>Graefe's archive for clinical and experimental ophthalmology</title><addtitle>Graefes Arch Clin Exp Ophthalmol</addtitle><addtitle>Graefes Arch Clin Exp Ophthalmol</addtitle><description>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.</description><subject>Animals</subject><subject>Antigens, Differentiation, T-Lymphocyte - genetics</subject><subject>Antigens, Differentiation, T-Lymphocyte - metabolism</subject><subject>Autoimmune Diseases - immunology</subject><subject>Autoimmune Diseases - pathology</subject><subject>Autoimmune Diseases - prevention & control</subject><subject>Basic Science</subject><subject>Blotting, Western</subject><subject>CD4-Positive T-Lymphocytes - immunology</subject><subject>Cell Culture Techniques</subject><subject>Disease Models, Animal</subject><subject>Down-Regulation - drug effects</subject><subject>Female</subject><subject>Flow Cytometry</subject><subject>Gene Silencing</subject><subject>Hypersensitivity, Delayed - immunology</subject><subject>Inducible T-Cell Co-Stimulator Protein</subject><subject>Injections</subject><subject>Lymphocyte Activation</subject><subject>Medicine</subject><subject>Medicine & Public Health</subject><subject>Ophthalmology</subject><subject>Peptide Fragments</subject><subject>Plasmids</subject><subject>Rats</subject><subject>Rats, Inbred Lew</subject><subject>Retinitis - immunology</subject><subject>Retinitis - pathology</subject><subject>Retinitis - prevention & control</subject><subject>Retinol-Binding Proteins</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>RNA Interference</subject><subject>RNA, Messenger - genetics</subject><subject>RNA, Messenger - metabolism</subject><subject>RNA, Small Interfering - administration & dosage</subject><subject>RNA, Small Interfering - genetics</subject><subject>Transfection</subject><subject>Uveitis - immunology</subject><subject>Uveitis - pathology</subject><subject>Uveitis - prevention & control</subject><subject>Vitreous Body</subject><issn>0721-832X</issn><issn>1435-702X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><recordid>eNp1kctq3DAUhk1paaZpH6CbIroIyUKtLvbIXobpLRAa6AWyExr5eNBgS66OlDYP1verzEwJFLo6unz_r3P0V9VLzt5wxtRbZKzmijLWUs6EpOxRteK1bKhi4vZxtWJKcNpKcXtSPUPcs4LLhj-tTnjHRSMUX1W_34WfnkbY5dEkFzwJA3G-z9ZtRyA2UExuWu5CJOdXm5uvF2R7X4gUzZ1LEcxYNnuwf7U4mXE5ShAHiM7vyJfPl-QcXSkXZB4NTq4nmOc5AiIgCX4XFgx-zYWfwKdiaXIKbpqyB5LvIERIzrvksBiTaBI-r54MZkR4cayn1fcP779tPtHrm49Xm8trauv1OtF2LS3rVc0kqFpakNADN1ZYOfRDv2162XZSDK2Add8M1krbWVVDC12nylrJ0-rs4DvH8CMDJj05tDCOxkPIqAVrWtU1TQFf_wPuQ46-9KaFZAXp2rpA_ADZGBAjDHouE5t4rznTS6D6EKgugeolUM2K5tXROG8n6B8UxwQLIA4Azst3Q3x4-f-ufwCqYrAA</recordid><startdate>20090601</startdate><enddate>20090601</enddate><creator>Hou, Yongsheng</creator><creator>Xing, Lin</creator><creator>Fu, Shaoying</creator><creator>Zhang, Xiaoning</creator><creator>Liu, Jingjing</creator><creator>Liu, Hongling</creator><creator>Lv, Bingjie</creator><creator>Cui, Hao</creator><general>Springer-Verlag</general><general>Springer Nature B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7TK</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>K9.</scope><scope>M0S</scope><scope>M1P</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7T5</scope><scope>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>RC3</scope></search><sort><creationdate>20090601</creationdate><title>Down-regulation of inducible co-stimulator (ICOS) by intravitreal injection of small interfering RNA (siRNA) plasmid suppresses ongoing experimental autoimmune uveoretinitis in rats</title><author>Hou, Yongsheng ; Xing, Lin ; Fu, Shaoying ; Zhang, Xiaoning ; Liu, Jingjing ; Liu, Hongling ; Lv, Bingjie ; Cui, Hao</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c466t-863c0d7403e743ce3ede1ac2c3fdfdb5d38932f82e6d5fcc3c9c74e8e9973c973</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>Animals</topic><topic>Antigens, Differentiation, T-Lymphocyte - genetics</topic><topic>Antigens, Differentiation, T-Lymphocyte - metabolism</topic><topic>Autoimmune Diseases - immunology</topic><topic>Autoimmune Diseases - pathology</topic><topic>Autoimmune Diseases - prevention & control</topic><topic>Basic Science</topic><topic>Blotting, Western</topic><topic>CD4-Positive T-Lymphocytes - immunology</topic><topic>Cell Culture Techniques</topic><topic>Disease Models, Animal</topic><topic>Down-Regulation - drug effects</topic><topic>Female</topic><topic>Flow Cytometry</topic><topic>Gene Silencing</topic><topic>Hypersensitivity, Delayed - immunology</topic><topic>Inducible T-Cell Co-Stimulator Protein</topic><topic>Injections</topic><topic>Lymphocyte Activation</topic><topic>Medicine</topic><topic>Medicine & Public Health</topic><topic>Ophthalmology</topic><topic>Peptide Fragments</topic><topic>Plasmids</topic><topic>Rats</topic><topic>Rats, Inbred Lew</topic><topic>Retinitis - immunology</topic><topic>Retinitis - pathology</topic><topic>Retinitis - prevention & control</topic><topic>Retinol-Binding Proteins</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>RNA Interference</topic><topic>RNA, Messenger - genetics</topic><topic>RNA, Messenger - metabolism</topic><topic>RNA, Small Interfering - administration & dosage</topic><topic>RNA, Small Interfering - genetics</topic><topic>Transfection</topic><topic>Uveitis - immunology</topic><topic>Uveitis - pathology</topic><topic>Uveitis - prevention & control</topic><topic>Vitreous Body</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hou, Yongsheng</creatorcontrib><creatorcontrib>Xing, Lin</creatorcontrib><creatorcontrib>Fu, Shaoying</creatorcontrib><creatorcontrib>Zhang, Xiaoning</creatorcontrib><creatorcontrib>Liu, Jingjing</creatorcontrib><creatorcontrib>Liu, Hongling</creatorcontrib><creatorcontrib>Lv, Bingjie</creatorcontrib><creatorcontrib>Cui, Hao</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Neurosciences Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central</collection><collection>ProQuest One Community College</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Immunology Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><jtitle>Graefe's archive for clinical and experimental ophthalmology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hou, Yongsheng</au><au>Xing, Lin</au><au>Fu, Shaoying</au><au>Zhang, Xiaoning</au><au>Liu, Jingjing</au><au>Liu, Hongling</au><au>Lv, Bingjie</au><au>Cui, Hao</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Down-regulation of inducible co-stimulator (ICOS) by intravitreal injection of small interfering RNA (siRNA) plasmid suppresses ongoing experimental autoimmune uveoretinitis in rats</atitle><jtitle>Graefe's archive for clinical and experimental ophthalmology</jtitle><stitle>Graefes Arch Clin Exp Ophthalmol</stitle><addtitle>Graefes Arch Clin Exp Ophthalmol</addtitle><date>2009-06-01</date><risdate>2009</risdate><volume>247</volume><issue>6</issue><spage>755</spage><epage>765</epage><pages>755-765</pages><issn>0721-832X</issn><eissn>1435-702X</eissn><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.</abstract><cop>Berlin/Heidelberg</cop><pub>Springer-Verlag</pub><pmid>19125271</pmid><doi>10.1007/s00417-008-1023-0</doi><tpages>11</tpages></addata></record> |
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| ispartof | Graefe's archive for clinical and experimental ophthalmology, 2009-06, Vol.247 (6), p.755-765 |
| issn | 0721-832X 1435-702X |
| language | eng |
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| subjects | Animals Antigens, Differentiation, T-Lymphocyte - genetics Antigens, Differentiation, T-Lymphocyte - metabolism Autoimmune Diseases - immunology Autoimmune Diseases - pathology Autoimmune Diseases - prevention & control Basic Science 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 Medicine Medicine & Public Health Ophthalmology Peptide Fragments Plasmids Rats Rats, Inbred Lew Retinitis - immunology Retinitis - pathology Retinitis - prevention & control Retinol-Binding Proteins Reverse Transcriptase Polymerase Chain Reaction RNA Interference RNA, Messenger - genetics RNA, Messenger - metabolism RNA, Small Interfering - administration & dosage RNA, Small Interfering - genetics Transfection Uveitis - immunology Uveitis - pathology Uveitis - prevention & control Vitreous Body |
| title | 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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