ABSTRACT
Increasing rates of life-threatening infections and decreasing susceptibility to antibiotics urge an effective vaccine targeting Staphylococcus aureus. Here we investigate the role of cellular immunity in FnBPA110-263 mediated protection in Staphylococcus aureus infection. This study revealed FnBPA110-263 broadly protected mice from seven FnBPA isotypes strains in the sepsis model. FnBPA110-263 immunized B-cell deficient mice were protected against lethal challenge, while T-cell deficient mice were not. Reconstituting mice with FnBPA110-263 specific CD4+ T-cells conferred antigen specific protection. In vitro assays indicated that isolated FnBPA110-263 specific splenocytes from immunized mice produced abundant IL-17A. IL-17A deficient mice were not protected from a lethal challenge by FnBPA110-263 vaccination. Moreover, neutralizing IL-17A, but not IFN-γ,reverses FnBPA110-263-induced protective efficacy in sepsis and skin infection model. These findings suggest that IL-17A producing Th17 cells play an essential role in FnBPA110-263 vaccine-mediated defense against S. aureus sepsis and skin infection in mice.
Subject(s)
Adhesins, Bacterial/immunology , Bacterial Vaccines/immunology , Sepsis/immunology , Staphylococcal Infections/immunology , Staphylococcus aureus/immunology , Animals , B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/microbiology , Immunity, Cellular/immunology , Interferon-gamma/immunology , Interleukin-17/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , Mice, SCID , Sepsis/microbiology , Th17 Cells/immunology , Th17 Cells/microbiology , Vaccination/methodsABSTRACT
Inhibitory signaling is crucial in the regulation of the cytotoxicity of natural killer (NK) cells. Here, we show that KIR2DL1, an inhibitory receptor of NK cells, associates with supervillin, an F-actin binding protein. Interaction of supervillin with KIR2DL1 is dependent on the KIR2DL1 receptor stimulation and requires the phosphorylation of tyrosines in both ITIM motifs. "Knockdown" of expression of supervillin by RNA interference (RNAi) restores the KIR2DL1-suppressed cytotoxicity of NK cells. Inhibition of supervillin by RNAi also enhances the polarization of cytolytic granules (both granzyme B and perforin) to the synapse formed between YTS-GFP-KIR2DL1 NK cells and 721.221-HLA-Cw4 target cells. Further study reveals that supervillin is required for KIR2DL1-mediated inhibition of Vav1 and ERK phoshorylation. Moreover, we have found that binding of supervillin with KIR2DL1 facilitates the recruitment of SHPs especially SHP-2 to KIR2DL1 receptor. Thus, our findings demonstrate that supervillin is a novel molecule that associates with KIR2DL1 receptor and regulates the inhibitory signaling in NK cells.
Subject(s)
Killer Cells, Natural/physiology , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Receptors, KIR2DL1/metabolism , Signal Transduction , Amino Acid Motifs , Cell Line, Transformed , Consensus Sequence , Cytotoxicity, Immunologic , HEK293 Cells , Humans , Killer Cells, Natural/immunology , Membrane Proteins/genetics , Microfilament Proteins/genetics , Phosphorylation , Protein Binding , Protein Transport , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , RNA Interference , Tumor Cells, CulturedABSTRACT
The adaptor protein paxillin plays an important role in cell migration. Although the c-Jun amino-terminal kinase (JNK) phosphorylation of paxillin on Ser 178 has been found to be critical for cell migration, the precise mechanism by which JNK regulates cell migration is still not very clear. Here, the migration of human corneal epithelial (HCE) cells was used to determine which signaling pathways are involved in EGF-induced paxillin phosphorylation. Paxillin was phosphorylated on Tyr 31 and Tyr 118 after induction of migration by EGF in HCE cells. Specific inhibition of JNK activation by inhibitor SP600125 or overexpression of a dominant-negative JNK mutant not only blocked EGF-induced cell migration, but also eliminated tyrosine phosphorylation of paxillin on Tyr 31 and Tyr 118. HCE cells overexpressing paxillin-S178A mutant also exhibited lower mobility, and reduced phosphorylation of Tyr 31 and Tyr 118. However, paxillin-S178A-inhibited cell migration can be rescued by overexpression of paxillin-Y31E/Y118E mutant. Importantly, inhibition of JNK by SP600125 or overexpression of paxillin-S178A mutant prevented the association of FAK with paxillin. Taken together, these results suggest that phosphorylation of paxillin on Ser 178 by JNK is required for the association of paxillin with FAK, and subsequent tyrosine phosphorylation of paxillin.
