April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Therapeutic Targeting of STAT3 Pathways of Macrophage and Th17 cells
Author Affiliations & Notes
  • Y. Lee
    Molecular Immunology, NEI/NIH, Bethesda, Maryland
  • C. E. Egwuagu
    Molecular Immunology, NEI/NIH, Bethesda, Maryland
  • Footnotes
    Commercial Relationships  Y. Lee, None; C.E. Egwuagu, None.
  • Footnotes
    Support  None.
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 821. doi:
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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)

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Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: signal transduction • immunomodulation/immunoregulation • inflammation 

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