Topical antisense-oligonucleotides targeting IFN-gamma mRNA improve incidence and severity of herpetic stromal keratitis by cytokine specific and sequence unspecific effects.
BACKGROUND
Corneal infection with herpes simplex virus-1 (HSV) can cause an inflammatory eye disease termed herpetic stromal keratitis (HSK). Interferon-gamma (IFN-gamma) is known to be involved in the development of this disease. In this study, antisense oligonucleotides targeting IFN-gamma mRNA (IFN-gamma-ASON) were investigated for their effects in experimental HSK.


METHODS
Splenic cells were used to examine the efficacy of IFN-gamma-ASON to decrease IFN-gamma- release into the cell culture supernatants as measured by ELISA. Mice were corneally infected with 10(5) PFU HSV, and IFN-gamma-ASON were given subepithelially. Alternatively, mice were infected without any further treatment, received only buffer, or received control oligonucleotides (CON) to observe substance specific effects. The animals were followed up clinically for the signs of herpetic keratitis. On days 14 and 28 post infection (p.i.), animals were sacrificed, and eyes were collected for histological analysis. On day 7 p.i., infectious virus particles in the eyes were determined by a plaque assay.


RESULTS
While IFN-gamma-ASON diminished the content of IFN-gamma in a concentration-dependent manner in vitro, CON showed no significant effects. Whereas buffer-treated and only infected mice showed severe necrotizing keratitis on day 14 p.i., this was abolished after treatment with IFN-gamma-ASON, even after 28 and 52 days. CON-treated mice also showed an improved HSK on day 14, but not on day 28. The incidence of the disease was also clearly diminished after treatment with IFN-gamma-ASON at all time points examined. The number of inflammatory cells in both the central and the peripheral cornea were strongly reduced after the application of IFN-gamma-ASON as compared to the controls. In contrast, the infectious viral particles in eyes at day 7 p.i. did not differ between the four groups.


CONCLUSIONS
Topical treatment with IFN-gamma-ASON induced a long-term improvement of the course and the incidence of HSK in the murine model. IFN-gamma seems to be involved in a proinflammatory manner during the pathogenesis of HSK, while the antiviral defense against HSV was not affected by this topical cytokine inhibition. Unspecific CON induced a transient and cytokine independent improvement of HSK.
Wasmuth
Susanne
S
Ophtha-Lab, Department of Ophthalmology, St. Franziskus Hospital, Hohenzollenring 74, 48145, Muenster, Germany.
Bauer
Dirk
D
Steuhl
Klaus-Peter
KP
Heiligenhaus
Arnd
A
D000287:Administration, Topical; D000818:Animals; D003319:Corneal Stroma / Q000187:drug effects* / Q000276:immunology / Q000821:virology; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D018390:Gene Targeting; D018259:Herpesvirus 1, Human / Q000502:physiology; D006968:Hypersensitivity, Delayed / Q000276:immunology; D007371:Interferon-gamma / Q000235:genetics* / Q000378:metabolism; D016849:Keratitis, Herpetic / Q000188:drug therapy* / Q000276:immunology / Q000821:virology; D051379:Mice; D008807:Mice, Inbred BALB C; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage*; D012333:RNA, Messenger / Q000235:genetics*; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D007371:Interferon-gamma