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S. Saraswathy, N. A. Rao; Immune Mechanisms Leading to Photoreceptor Mitochondrial Oxidative Stress During Early EAU. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3894.
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We have previously shown photoreceptor mitochondrial oxidative stress leads to marked upregulation of αA crystallin in the photoreceptor inner segments during early EAU before leukocyte infiltration in the retina and/or uvea. The crystallin upregulation is a superb marker for mitochondrial oxidative stress. In this study, we investigated the cellular and immunologic events that initiate this oxidative stress using T bet KO, TNF-α KO, iNOS KO and B10.RIII mice.
EAU was induced in C57BL/6 (Wild Type; WT), T-bet KO, TNF-α KO, iNOS KO and B10.RIII, mice with IRBP. WT mice injected with CFA and non-immunized mice served as controls. Gene expression levels of iNOS, αA crystallin and IL17 in the retinas during early EAU was quantitated by real-time PCR. RNA isolated from the retinas of B10.RIII mice was also subjected to T cell and immune response microarray analysis and q-PCR, and those from TNF-α KO mice was subjected to apoptosis PCR array. Apoptosis was studied in the iNOS KO retinas by TUNEL assay.
mRNA expression of iNOS, αA crystallin and IL-17 were elevated in the WT EAU mice compared to the controls. In contrast, there was only 50% increase in their expression in the T-bet KO mice compared to the WT animals. Microarray analysis revealed upregulation of T cell activating genes, T cell markers, Th1 transcription factor and the Th1 and Th17 related cytokines mainly, TNF-α, TNF receptor 1 (TNF-R1), IL-17, IL-18, IL-1, IL-6, and IL-15. The cytokine genes that are reported to upregulate iNOS, specifically, TNF-α, IL-12, IL-1, IL-17, IL-18 and IL-15, were elevated during early EAU. iNOS was not upregulated in TNF-α KO mice and there was no change in αA crystallin gene expression in either TNF-α KO or iNOS KO mice during early EAU. PCR array revealed decreased expression of apoptotic genes in TNF-α KO mice compared to WT animals. Apoptotic cells were absent in the retinas of iNOS KO mice
Upregulation of iNOS and αA crystallin in the T-bet KO mice with EAU was reduced more than 50% compared to the WT with EAU, and increased expression of both Th1 and Th17 related cytokines like TNF-α, IL-12 and IL-17, indicate that both Th1 and Th17 responses may play a role in inducing oxidative stress during early EAU. TNF-α plays major role in the generation of iNOS and later in the induction of mitochondrial stress as detected by upregulated αA crystallin levels. TNF-α also seem to be modulating the genes in the apoptotic signaling pathway in the earlier stages of EAU. Deletion of iNOS gene attenuated induction of αA crystallin and apoptosis suggesting that upregulation of αA crystallin clearly depends on prior induction of iNOS
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