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Molecular Pathology and Immunology Program
M.S. 2003, the Fourth Military Medical University, China
M.D. 2000, the Fourth Military Medical University, China
Thesis Advisor: Patricia Fitzgerald-Bocarsly, Ph.D.
Department of Pathology and Laboratory Medicine
Thursday, January 19, 2012
1:30 P.M., C-555
Plasmacytoid dendritic cells (PDC) play important roles in innate immunity in that they produce a large amount of type I IFN in response to virus and synthetic TLR7 and 9 agonists such as CpG ODNs. CpGA is a potent IFN-á inducer, whereas CpGB induces PDC maturation but not type I IFN expression. We were interested in investigating how NF-êB regulates IFN-á production in PDC. Using SN50, a specific inhibitor of NF-êB nuclear translocation, we found that CpGA-induced IFN-á production was inhibited. Although CpGB did not by itself induce IFN-á production, SN50 conferred upon CpGB the ability to induce a large amount of IFN-á production. Using ImageStream imaging flow cytometry, we found that both CpGA and CpGB-induced NF-êB nuclear translocation was inhibited by SN50. In addition, CpGA-induced IRF7 (a transcription factor required for IFN-á production) nuclear translocation was inhibited by SN50. In contrast, although CpGB did not induce IRF7 translocation, SN50 conferred upon CpGB the ability to induce IRF7 translocation. Similar discoveries were found with NBD peptide, another NF-êB inhibitor, but not with a non-peptide NF-êB inhibitor, JSH-23. Concurrent published literature indicated that peptides like HMGB1 and LL37 can also confer upon CpGB the ability to induce IFN-á and both are cationic peptides, which form complexes with CpGB. We therefore designed a scrambled peptide (SP), which keeps the cationic charge but eliminates the core structure of SN50 making it unable to inhibit NF-êB activity. Interestingly, although SP did not inhibit CpGB-induced NF-êB nuclear translocation, it still conferred upon CpGB the ability to induce IFN-á production. These results suggested that the ability of SN50 and NBD peptides to make CpGB able to induce IFN-á is not due to NF-êB inhibition, but, rather, by forming complexes with CpGB. Indeed, gel retardation assays showed that CpGB did form complexes with SN50 and SP. Altogether, these results suggested that it is the cationic property of peptides SN50, SP, and NBD that confers upon CpGB the ability to induce IFN-á production by forming higher order structures similar to those formed naturally by CpGA.
It has been reported that TLR9 is recruited to endosomes upon stimulation in a TLR9-transfected cell line. But there has been no report about TLR9 trafficking in human PDC. By ImageStream flow cytometry, we found that TLR9 constitutively resided in both early and late endosomes in human PDC, and more TLR9 was recruited from ER to endosomes upon stimulation with CpGA and HSV. It has been shown that TLR9 cleavage is required for its function in murine PDC. We found that both HSV and CpGA-induced IFN-á production was inhibited by a pan-cathepsin inhibitor, which inhibits TLR9 cleavage by inhibiting cysteine protease activity, suggesting that TLR9 cleavage is required for its function in human PDC.
Together, the findings in this dissertation contribute to our understanding of regulation of PDC activation. Such understanding of the mechanisms of how IFN-á is produced and regulated in PDC, may lead to novel strategies for identification of therapeutic targets for a variety of diseases, such as autoimmune diseases, cancer and AIDS.