Induction of E6/E7 Expression in Cottontail Rabbit Papillomavirus Latency Following UV Activation

AbstractLatent human papillomavirus (HPV) infections are widespread in the genital and respiratory tracts and are a source of recurrent disease. This study used a cottontail rabbit papillomavirus (CRPV) model to determine the presence of E1, E6, and E7 transcripts in latent infection and to determine the temporal change in transcripts following UV activation. We found E1 transcripts in all latently infected sites but no detectable E6 and E7 transcripts, consistent with our earlier studies of HPV6/11 latency. These results suggest that this transcription pattern is broadly characteristic of latent papillomavirus infections. E6/E7 transcripts were detectable within 1 week of irradiation, with maximal induction (approximately 40% of sites) at 2 weeks postirradiation. Papillomas were induced in approximately 26% of irradiated sites after a 3- to 5-week lag. Sites that did not form papillomas by 3 months after irradiation were CRPV DNA positive but E6/E7 RNA negative. Thus, only a subset of latent infections can be induced to express E6/E7 transcripts and form papillomas. We propose that CRPV can be used to study the molecular processes regulating papillomavirus activation

Extracellular vesicles produced by primary human keratinocytes in response to TLR agonists induce stimulus-specific responses in antigen-presenting cells.

Cells can communicate through the extracellular vesicles (EVs) they secrete. Pathogen associated molecular patterns (PAMPs), alter the biophysical and communicative properties of EVs released from cells, but the functional consequences of these changes are unknown. Characterization of keratinocyte-derived EVs after poly(I:C) treatment (poly(I:C)-EVs) showed slight differences in levels of EV markers TSG101 and Alix, a loss of CD63 and were positive for autophagosome marker LC3b-II and the cytokine IL36γ compared to EVs from unstimulated keratinocytes (control-EVs). Flagellin treatment (flagellin-EVs) led to an EV marker profile like control-EVs but lacked LC3b-II. Flagellin-EVs also lacked IL-36γ despite nearly identical intracellular levels. While poly(I:C) treatment led to the clear emergence of a > 200 nm diameter EV sub-population, these were not found in flagellin-EVs. EV associated IL-36γ colocalized with LC3b-II in density gradient analysis, equilibrating to 1.10 g/mL, indicating a common EV species. Poly(I:C), but not flagellin, induced intracellular vesicles positive for IL-36γ, LC3b-II, Alix and TSG101, consistent with fusion of autophagosomes and multivesicular bodies. Simultaneous rapamycin and flagellin treatment induced similar intracellular vesicles but was insufficient for the release of IL-36γ+/LC3b-II+ EVs. Finally, a qRT-PCR array screen showed eight cytokine/chemokine transcripts were altered (p < 0.05) in monocyte-derived Langerhans cells (LCs) when stimulated with poly(I:C)-EVs while three were altered when LCs were stimulated with flagellin-EVs compared to control-EVs. After independent confirmation, poly(I:C)-EVs upregulated BMP6 (p = 0.035) and flagellin-EVs upregulated CXCL8 (p = 0.005), VEGFA (p = 0.018) and PTGS2 (p = 0.020) compared to control-EVs. We conclude that exogenous signals derived from pathogens can alter keratinocyte-mediated modulation of the local immune responses by inducing changes in the types of EVs secreted and responses in antigen presenting cells

Immune Dysregulation in Patients Persistently Infected with Human Papillomaviruses 6 and 11

Human Papillomaviruses (HPVs) 6 and 11 are part of a large family of small DNA viruses, some of which are commensal. Although much of the population can contain or clear infection with these viruses, there is a subset of individuals who develop persistent infection that can cause significant morbidity and on occasion mortality. Depending on the site of infection, patients chronically infected with these viruses develop either recurrent, and on occasion, severe genital warts or recurrent respiratory papillomas that can obstruct the upper airway. The HPV-induced diseases described are likely the result of a complex and localized immune suppressive milieu that is characteristic of patients with persistent HPV infection. We review data that documents impaired Langerhans cell responses and maturation, describes the polarized adaptive T-cell immune responses made to these viruses, and the expression of class select II MHC and KIR genes that associate with severe HPV6 and 11 induced disease. Finally, we review evidence that documents the polarization of functional TH2 and T-regulatory T-cells in tissues persistently infected with HPV6 and 11, and we review evidence that there is suppression of natural killer cell function. Together, these altered innate and adaptive immune responses contribute to the cellular and humoral microenvironment that supports HPV 6 and 11-induced disease