ABSTRACT
Dendritic cells (DCs) must achieve a critical balance between activation and tolerance, a process influenced by cytokines and growth factors. IL-10, which transduces signals through Stat3, has emerged as one important negative regulator of DC activation. To directly examine the role Stat3 plays in regulating DC activity, the Stat3 gene was targeted for deletion with a CD11c-cre transgene. Stat3 CKO mice developed cervical lymphadenopathy as well as a mild ileocolitis that persisted throughout life and was associated with impaired weight gain. Consistent with this, Stat3-deficient DCs demonstrated enhanced immune activity, including increased cytokine production, Ag-dependent T-cell activation and resistance to IL-10-mediated suppression. These results reveal a cell-intrinsic negative regulatory role of Stat3 in DCs and link increased DC activation with perturbed immune homeostasis and chronic mucosal inflammation.
Subject(s)
Dendritic Cells/immunology , Dendritic Cells/metabolism , STAT3 Transcription Factor/metabolism , Animals , CD11c Antigen/genetics , CD11c Antigen/metabolism , Cells, Cultured , Crohn Disease/genetics , Crohn Disease/metabolism , Crohn Disease/pathology , Cytokines/blood , Cytokines/metabolism , Dendritic Cells/drug effects , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Interferon-gamma/blood , Interferon-gamma/metabolism , Interleukin-10/pharmacology , Lymphatic Diseases/genetics , Lymphatic Diseases/metabolism , Lymphatic Diseases/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Mice, Transgenic , Reverse Transcriptase Polymerase Chain Reaction , STAT3 Transcription Factor/genetics , Tumor Necrosis Factor-alpha/blood , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The human T-cell leukemia virus type 1 (HTLV-1) Gag polyprotein contains two adjacent proline-rich motifs (sequence PPPYVEPTAP) in the C terminus of the matrix domain [corrected]. Proline-to-alanine mutations were introduced into either or both motifs of HTLV-1 to determine the effect on the release of HTLV-1 virus-like particles from 293T cells. The release of both single mutants was significantly reduced, whereas a double mutation in both motifs abolished the release of the HTLV-1 particles. Two-hybrid and in vitro binding assays showed that the HTLV-1 Gag polyprotein binds both Tsg101 and Nedd4 proteins. The interaction with HTLV-1 Gag required the central WW domain of Nedd4 and the ubiquitin enzyme variant (UEV) domain of Tsg101. We expressed various fragments of Nedd4 and Tsg101 proteins in 293T cells and tested for their ability to interfere with virion release mediated by the HTLV-1 Gag-Pro polyprotein. Fragments consisting of the N-terminal UEV domain of Tsg101 and the central WW and C-terminal Hect domains of Nedd4 protein all caused transdominant inhibition of HTLV-1 particle release. Similarly, inhibition of the proteasome significantly decreased HTLV-1 particle release. Furthermore, the WW domain overexpression caused an early arrest of HTLV-1 particle morphogenesis before the membrane is deformed into the typical half-shell structure. This result suggests that Nedd4 is involved early in budding of HTLV-1.