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"An alternative, IRF7-independent pathway of Type I IFN induction in primary human plasmacytoid dendritic cells"

by
Mahwish Natalia
Molecular Pathology and Immunology Program
M.A. 2006, Columbia University, NY
B.S. 2003, Marymount Manhattan College, NY


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

Monday, June 8, 2015
10:00 A.M., Room B610 Lecture Hall 4


Abstract

Human plasmacytoid dendritic cells (pDC) respond to influenza virus A (Flu), human immunodeficiency virus type 1 (HIV-1) and imidazoquinolines through toll-like receptor (TLR)-7, and herpes simplex virus type 1 (HSV) and CpGA through TLR9, by producing type I and type III interferons (IFNs) and pro-inflammatory cytokines. Imidazoquinolines are small molecules that are known for their anti-viral properties and immune adjuvancy and exert their effects via signaling through TLR7. Although Imiquimod is thought to induce IFN- production via the TLR7 pathway in pDCs, the exact signaling mechanism is unknown. Interestingly, imidazoquinolines have also been shown to inhibit TLR9-induced cytokine production, which has led some to suggest that the TLR7 signaling pathway dominates over the TLR9 pathway. We investigated the mechanisms by which the imidazoquinoline, 3M003, induces type I IFN production in primary human pDCs from human peripheral blood while inhibiting both TLR7 and -9 induced cytokine production. Using flow cytometry and ELISA assays, we found that 3M003 induced IFN-, IFN- and TNF- was a very early but short-lived response compared to the response to Flu or HSV. 3M003 required the induction of actin polymerization, endosomal acidification and TLR7 signaling to trigger cytokine production in pDCs as inhibitors of these pathways prevented 3M003-mediated cytokine production. Interestingly, 3M003 not only inhibited HSV and CpGA, but also Flu and HIV-induced cytokine production in pDC when added within the first four hours of virus stimulation. Interferon regulatory factor 7 (IRF7) is the major transcription factor shown to be involved in type I IFN production. Similar to Flu and HSV, 3M003 enhanced IRF7 serine 477/479 phosphorylation (indicative of its activation), but in contrast to Flu and HSV, which induce robust IRF7 translocation to the nucleus, 3M003 stimulation of pDC resulted in very little translocation of IRF7 over a range of times studied. We therefore investigated the translocation of IRF5, another transcription factor that is constitutively expressed in human pDC and has sometimes been implicated in the induction of type I IFN in model systems, but whose role in IFN- production has not been assessed in primary pDC. While HSV and Flu induced both IRF5 and IRF7 translocation, 3M003 only translocated IRF5. Similar results were obtained with R848, another member of the imidazoquinoline family. Flu and 3M003, but not CpGA-induced IRF5 and IRF7 translocations were inhibited with a TLR7 antagonist, indicating that Flu and 3M003-mediated activation of IRF5 and IRF7 is downstream of the TLR7 signaling pathway. Dual stimulation of Flu or HSV with 3M003 resulted in the levels of IFN-, phosphorylated IRF7 and nuclear translocation of IRF7 similar to those of 3M003 alone. 3M003 co-stimulated with HSV or Flu resulted in robust IRF5 translocation, similar to that seen with 3M003 stimulation alone. Activation of the PI3-kinase (PI3-K) pathway has been shown to be necessary for IRF7 translocation in pDC. We found that the PI3-K inhibitor Wortmannin was able to inhibit HSV and Flu-induced IFN- production, but did not affect 3M003-induced IFN- production, demonstrating that 3M003 induced IFN- production was not dependent on the PI3-K pathway. Taken together, these results indicate a novel signaling pathway in pDCs wherein the TLR7 agonist 3M003 stimulates IFN- production in an IRF7 and PI3-K-independent, IRF5-dependent manner resulting in the rapid, but low total production of IFN-. Moreover, triggering of this early, IRF5-dependent pathway by 3M003 was sufficient to derail the IRF7-dependent responses of pDC to viruses. This finding not only indicates that novel signaling mechanisms within the IFN induction pathway in pDC are induced by 3M003, but may also have important implications in the use of imidazoquinolines as therapeutic agents in human disease.


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