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Jing Deng
Molecular Pathology and Immunology Program
B.S. 2005, Wuhan University, China

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

Wednesday, November 7, 2012
10:00 A.M., Rosemary Gellene Room, MSB B515


Human plasmacytoid dendritic cells (pDC) are the most potent cells in the body to secrete large amounts of type I interferon (IFN-) in response to viruses and synthetic TLR7 or -9 agonists. Recent studies in mice have implicated autophagy as a player in cytokine production to infectious virus. However, the mechanisms of virus trafficking and virus-stimulated IFN- production without infection in the antiviral responses of human pDCs remain unclear. In order to understand the subcellular trafficking of viral particles in human pDC and the role of autophagy in this process, we investigated these intracellular events after stimulating human pDC with different viral stimuli as well as TLR7 and -9 agonists. We observed that the formation of double-membrane autophagosomes in pDC after HSV stimulation via transmission electron microscopy. Stimulation of pDC with viruses led to the rapid induction of LC3 aggregation, a hallmark of autophagosome formation.Interestingly, the prevention of autophagosome formation at an early time point by both class I and III PI3K inhibitors wortmannin, 3-methyladenine (3MA) and LY294002 was able to suppress virus-induced IFN- production at later time points. However, the uptake of herpes simplex virus expressing GFP (HSV-GFP) was not inhibited by the PI3K inhibitors. In contrast, an accumulation of the internalized HSV-GFP was observed, suggesting the formation of autophagsomes was specifically blocked by the PI3K inhibitors and this formation might be required for virus-induced IFN- production. siRNA knockdown of the autophagy genes Beclin1 and Atg7 significantly suppressed virus-induced IFN- production and LC3 aggregation, indicating autophagy is required for virus-induced IFN- production in pDCs.
To further trace the events related to virus-induced formation of autophagosomes, we investigated the colocaliztion of GFP-HSV viral particles, the Rab5+ early endosome compartment and LC3+ autophagosome compartment. Surprisingly, we found the Rab5+ early endosome compartment was colocalized with LC3+ autophagosome compartment in pDC at an early time point after viral stimulation, indicating the amphisomes, which have been characterized as the autophagic vacuoles formed upon fusion between autophagosomes and endosomes, were induced by viral stimulation. Moreover, using chloroquine, an inhibitor of endosomal fusion and acidification, the formation of amphisomes was significantly inhibited; however, the internalized GFP-HSV particles were accumulated without being able to be further enzymatically digested in the endosome. As a consequence, IFN- production was indeed inhibited with chloroquine treatment. We further investigated the interactions between early endosomes, autophagosomes and TLR9 after HSV stimulation using imaging flow cytometry; HSV-GFP particles were internalized into early endosomes by endocytosis and were subsequently transported to amphisomes. Concurrent with the trafficking of HSV-GFP, TLR9 rapidly redistributed to HSV-accumulating amphisomes by colocalizing with LC3 within 30min for ligand binding and signal transduction.
Collectively, our studies unveil a previously unknown mechanism of autophagy and amphisome formation in virus-induced IFN- production possibly involving the recognition of virus and the recruitment of TLR9 to sites of virus uptake in human pDC

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