Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Jan;85(2):895-904.
doi: 10.1128/JVI.01007-10. Epub 2010 Oct 27.

Activation of plasmacytoid dendritic cells by Kaposi's sarcoma-associated herpesvirus

John A West  1 , Sean M Gregory, Vijay Sivaraman, Lishan Su, Blossom Damania
Affiliations

Activation of plasmacytoid dendritic cells by Kaposi's sarcoma-associated herpesvirus

John A West et al. J Virol. 2011 Jan.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with multiple human malignancies, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Following primary infection, KSHV typically goes through a brief period of lytic replication prior to the establishment of latency. Plasmacytoid dendritic cells (pDCs) are the major producers of type 1 interferon (IFN), primarily in response to virus infection. Toll-like receptors (TLRs) are key components of the innate immune system, and they serve as pathogen recognition receptors that stimulate the host antiviral response. pDCs express exclusively TLR7 and TLR9, and it is through these TLRs that the type 1 interferon response is activated in pDCs. Currently, it is not known whether KSHV is recognized by pDCs and whether activation of pDCs occurs in response to KSHV infection. We now report evidence that KSHV can infect human pDCs and that pDCs are activated upon KSHV infection, as measured by upregulation of CD83 and CD86 and by IFN-α secretion. We further show that induction of IFN-α occurs through activation of TLR9 signaling and that a TLR9 inhibitor diminishes the production and secretion of IFN-α by KSHV-infected pDCs.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Purity of pDCs isolated from human blood. We show a representative data set on the purities we were able to achieve upon isolation of pDCs from human donors. Purification was carried out as described in Materials and Methods, using magnetic bead-based separation. Purified pDCs were stained with BDCA-2-FITC (CD303) either alone (A) or in combination with CD123-PE (B) to determine cell purity. Data analysis was performed using Summit v4.3.
FIG. 2.
FIG. 2.
CD83 activation at 16 h in KSHV-infected pDCs. pDCs were isolated from four different donors, and cells were either mock infected or infected with KSHV. Cells were harvested at 16 h postinfection and stained using an anti-CD83-APC antibody. Flow cytometry was performed on a Miltenyi MACSQuant analyzer. The data were analyzed using Summit v4.3.
FIG. 3.
FIG. 3.
CD86 activation at 16 h in KSHV-infected pDCs. pDCs were isolated from four different donors. Cells were infected with KSHV or mock infected. Cells were harvested at 16 h postinfection and stained using an anti-CD86-APC antibody. Flow cytometry was performed on a Miltenyi MACSQuant analyzer. The data were analyzed using Summit v4.3.
FIG. 4.
FIG. 4.
CD83 and CD86 activation 16 h after infection of pDCs with KSHV. Cells were either mock infected, infected with wild-type (WT) KSHV, or infected with UV-inactivated KSHV (UV-KSHV). Cells were harvested at 16 h postinfection and stained with either CD83-APC or CD86-APC antibody. Flow cytometry was performed on a Miltenyi MACSQuant analyzer. The data were analyzed using Summit v4.3.
FIG. 5.
FIG. 5.
KSHV infection of pDCs. (A) Mock- or KSHV-infected live pDCs were imaged using confocal microscopy to identify KSHV-infected cells. Cells expressing GFP were detected only in KSHV-infected pDCs, whereas no GFP was detected in the mock-infected cells. Images were taken on a Zeiss LSM5 Pa laser scanning microscope and processed using the Zeiss LSM Image browser. (B) Percent GFP expression in mock-infected pDCs versus KSHV-infected pDCs at 16 h postinfection. Flow cytometry of mock- and KSHV-infected cells. (C) Orf57 transcription in KSHV-infected pDCs. Cell pellets were processed for RNA and reverse transcribed to cDNA. RT-PCR was performed using primers for the viral early lytic gene Orf57. KSHV Orf57 transcription was detected only in KSHV-infected cells, not in mock-infected cells. Controls included RT-PCR of β-actin transcript levels. +RT, reverse transcription of the isolated RNA; −RT, isolated RNA was added to the RT-PCR mix to serve as a control for genomic DNA contamination; NT, no template was added to the RT-PCR mix to serve as a negative control for the primer sets. (D) Orf73 transcription in KSHV-infected pDCs. Analysis was performed as described for panel B. Controls included RT-PCR of GAPDH transcript levels.
FIG. 6.
FIG. 6.
KSHV infection of pDCs induces secretion of IFN-α. (A) IFN-α secretion was measured at different time points post-KSHV infection. Supernatants from infected pDCs were collected at the indicated times postinfection and analyzed for the presence of IFN-α by ELISA. Mock-infected pDCs were used as the control. (B) Isolated pDCs were subjected to mock infection, WT KSHV infection, or infection with UV-KSHV at the indicated time points. Supernatants were analyzed for the presence of IFN-α by ELISA.
FIG. 7.
FIG. 7.
TLR9 inhibition blocks IFN-α secretion following KSHV infection. (A) pDCs were treated with a TLR9-inhibitory oligonucleotide (G-ODN) at a 25 μM final concentration simultaneously with either KSHV or 10 μM CpG DNA (as a positive control). Infections were carried out as described in Materials and Methods. Supernatants were harvested at 16 h postinfection and were analyzed for IFN-α production by ELISA. IFN-α secretion is shown for cells that were either mock infected, infected with KSHV, or treated with CpG (TLR9 agonist) in the presence or absence of the TLR9 inhibitor G-ODN. (B) pDCs were obtained from 2 different donors and were either mock infected or infected with KSHV in the presence or absence of the TLR9 inhibitor. IFN-α secretion was measured at 16 and 45 h post-KSHV infection. (C) DNase treatment of purified KSHV intact virions does not reduce IFN-α levels following infection. Purified KSHV was treated for 10 min at 37°C with 0.1 U of DNase I. pDCs were infected with either WT or DNase-treated KSHV, and supernatants were collected and analyzed for the presence of IFN-α.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Akira, S., and H. Hemmi. 2003. Recognition of pathogen-associated molecular patterns by TLR family. Immunol. Lett. 85:85-95. - PubMed
    1. Akira, S., and S. Sato. 2003. Toll-like receptors and their signaling mechanisms. Scand. J. Infect. Dis. 35:555-562. - PubMed
    1. Akira, S., and K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4:499-511. - PubMed
    1. Bauer, S., C. J. Kirschning, H. Hacker, V. Redecke, S. Hausmann, S. Akira, H. Wagner, and G. B. Lipford. 2001. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc. Natl. Acad. Sci. U. S. A. 98:9237-9242. - PMC - PubMed
    1. Blasig, C., C. Zietz, B. Haar, F. Neipel, S. Esser, N. H. Brockmeyer, E. Tschachler, S. Colombini, B. Ensoli, and M. Sturzl. 1997. Monocytes in Kaposi's sarcoma lesions are productively infected by human herpesvirus 8. J. Virol. 71:7963-7968. - PMC - PubMed

Publication types

LinkOut - more resources