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"Title: Dendritic cell activation: i) Plasmacytoid and Myeloid DC:HIV-infected cell interactions and ii) Effects of HDAC inhibitors on Plasmacytoid Dendritic cells"

Dante B. Descalzi-Montoya
Molecular Pathology and Immunology Program
M.S. 2007, William Paterson University, NJ
B.A. 2004, Rutgers University and NJIT, NJ

Thesis Advisor: Patricia Fitzgerald-Bocarsly, Ph.D.
Department of Pathology and Laboratory Medicine

Thursday, August 21, 2014
1:00 P.M., MSB C-555


Dendritic cell activation by viruses and virus-infected cells is incompletely understood. The first project (Section 1) investigates mechanistic aspects of fusion and nibbling between, mDC and pDC, and HIV-1-infected target cells. The second project (Section 2) investigates the mechanism of inhibition of pDC function by histone deacetylase inhibitors (HDCAi).
Section 1:
It is well documented that pDC suffer a functional and numerical loss in the blood and tissues of HIV-1-infected individuals. Our group and others have proposed that one of the mechanisms of HIV-1-induced pDC depletion in vivo is the fusion between uninfected pDC and HIV-1-infected T cells. Previously, our group made in-vitro observations of pDC fusion with primary, acutely HIV-infected CD4+ T cells and chronically HIV-infected T cells. In the current study, we have delved into the mechanistic aspects of this process. First, we observed that mDC, like pDC, can also fuse with and nibble from chronically-infected T cells; a process that was insensitive to CCR5 and gp120 blockade, but sensitive to CXCR4 blockade. HIV-1 also induced fusion and nibbling between pDC and a HIV-infected pDC-like cell line, GEN2.2. Moreover, pDC:T cell fusion did not require endosomal acidification, was TLR-7 independent, did not require active HIV infection, but did require cytoskeleton integrity to occur. Moreover, pDC:T cell fusion products underwent apoptosis as evidenced by higher Annexin-V binding than pDC or T cells alone or pDC that had nibbled membrane material from infected T cells. Third, the effects of TLR7/9 activation of pDC on fusion showed that IFN- inducing ligands enhanced fusion, but decreased nibbling. TLR9-induced fusion enhancement was specifically blocked by A151 ODN, an inhibitor of TLR9 signaling. On the other hand, a maturation inducing TLR9 ligand (CpG-B) enhanced nibbling, but antagonized fusion. Moreover, exogenous IFN- treatment significantly increased fusion but decreased nibbling between activated pDC and HIV-1-infected T cells, which suggests that IFN- may play a critical role in fusion. Interestingly, A151 reduced IFN--induced fusion, suggesting an additional mechanism of inhibition by A151 besides the documented TLR9 blockade and further suggests a potential therapeutic use for this ODN. In conclusion, this study encompasses important mechanistic aspects of DC:T cell fusion and nibbling and highlights the significance of IFN- and TLR7/9 activation and HIV infection.
Section 2:
Histone deacetylase inhibitors (HDACi) are currently being considered for therapeutic use in the clinic due to their powerful ability to induce the activation of latent HIV and to ameliorate the symptoms of autoimmunity. However, HDACi also have non-specific effects on various aspects of the immune system that otherwise aid in fighting off infection. In this study we characterized the effects of HDACi on pDC function. We observed that treatment of human pDC with broad Class I/II HDACi TSA (Trichostatin A) and suberoylanilide hydroxamic acid (SAHA/Vorinostat), or Class I HDACi MS-275 strongly inhibited the induction of IFN- and TNF- upon stimulation with TLR7/9 synthetic and viral stimuli. This inhibitory effect by TSA, unlike SAHA, was not associated TLR7/9-induced IRF-7 protein upregulation during the first 6 hours of stimulation. In contrast, TSA was able to inhibit exogenous IFN--mediated upregulation of IRF-7. Treatment with TSA, SAHA, or MS-275 did not induce apoptosis or affected pDC numbers. Furthermore, TSA, but not MS-275, inhibited TLR7/9-induced expression of maturation and activation markers.TSA and SAHA effects on pDC function were associated with a decrease in the nuclear translocation of transcription factors IRF-7 and NF-қB p65, which was found to be partially caused by a decline in their phosphorylation by kinases. Importantly, TSA blocked the formation of HIV-1-induced pDC:T cell fusion in vitro. In conclusion, inhibition of TLR7/9 and IFN- receptor signaling, by TSA and SAHA, can explain HDACi effects on TLR7/9-induced cytokine production, maturation, and activation. These findings could potentially present a problem for the therapeutic use of HDACi, but on the other hand can help thwart HIV spread and transmission, which prompts for development of more specific HDACi that should target HIV latency with minimal effect on the immune system.

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