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Y. Lee, C. E. Egwuagu; Therapeutic Targeting of STAT3 Pathways of Macrophage and Th17 cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):821.
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© ARVO (1962-2015); The Authors (2016-present)
Mice with targeted-deletion of STAT3 in CD4+ T-cells do not develop EAU or EAE because of their inability to generate pathogenic Th17 cells. STAT3-deficient T cells do not enter the eye or brain suggesting that STAT3 is required for T cell trafficking into CNS. STAT3 is therefore an attractive therapeutic target that can be used to inhibit uveitis. SOCS3 is a potent inhibitor of STAT3 pathways and can be used for therapeutic targeting of STAT3. However, a major huddle to the use of SOCS3 is the difficulty of intracellular delivery of SOCS3. Recently, it was shown that fusion of any protein to a hydrophobic 12 AA sequence of the signal peptide of the Karposi FGF4 protein (AAVLLPVLLAAP) enhances penetration of the recombinant protein into cells. In this study, we have generated a recombinant SOCS3 protein that is fused to the membrane translocating sequence (MTS) and examined whether it can be used to target STAT3 pathways of macrophage and Th17 cells.
The mouse SOCS3 cDNA was fused to the MTS sequence inserted into a bacterial expression vector containing N-terminal His tag (six histidine residues). The recombinant SOCS3-MTS fusion protein was purified using Ni-column. Macrophage cells were stimulated with cytokine and we examined the effects of SOCS3-MTS on STAT3 pathway by Western blotting. Naïve CD4+ T cells were isolated from the spleen and lymph nodes of C56BL/6 mice and stimulated by T Cell receptor cross-linking under Th17 polarization condition (IL-6, TGF-β, anti-IFN-γ and anti-IL-4 antibodies) in the presence or absence of SOCS3-MTS. We then examined the effects of SOCS3-MTS on T cell differentiation by flow cytometry.
We demonstrate that the SOCS3-MTS protein translocates into the cytoplasm of the macrophage cell. We also show that the SOCS3-MTS protein reduced pSTAT3 level in macrophage cells that were stimulated with IL-6. We further show that treatment of SOCS3-MTS protein under Th17 polarizing condition inhibits Th17 cell differentiation.
Our data shows that SOCS3-MTS inhibits activation of STAT3 pathway in macrophage and inhibits Th17 cell differentiation. Thus, SOCS3-MTS might be used as a therapeutic molecule to inhibit anterior uveitis caused by innate immune cells and also posterior uveitis caused by Th17 cells.
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