TLR-9 Contributes to the Antiviral Innate Immune Sensing of Rodent Parvoviruses MVMp and H-1PV by Normal Human Immune Cells

<div><p>The oncotropism of Minute Virus of Mice (MVMp) is partially related to the stimulation of an antiviral response mediated by type-I interferons (IFNs) in normal but not in transformed mouse cells. The present work was undertaken to assess whether the oncotropism displayed against human cells by MVMp and its rat homolog H-1PV also depends on antiviral mechanisms and to identify the pattern recognition receptor (PRR) involved. Despite their low proliferation rate which represents a drawback for parvovirus multiplication, we used human peripheral blood mononuclear cells (hPBMCs) as normal model specifically because all known PRRs are functional in this mixed cell population and moreover because some of its subsets are among the main IFN producers upon infections in mammals. Human transformed models consisted in lines and tumor cells more or less permissive to both parvoviruses. Our results show that irrespective of their permissiveness, transformed cells do not produce IFNs nor develop an antiviral response upon parvovirus infection. However, MVMp- or H-1PV-infected hPBMCs trigger such defense mechanisms despite an absence of parvovirus replication and protein expression, pointing to the viral genome as the activating element. Substantial reduction of an inhibitory oligodeoxynucleotide (iODN) of the latter IFN production identified TLR-9 as a potential PRR for parvoviruses in hPBMCs. However, neither the iODN treatment nor an antibody-induced neutralization of the IFN-triggered effects restored parvovirus multiplication in these cells as expected by their weak proliferation in culture. Finally, given that a TLR-9 activation could also not be observed in parvovirus-infected human lines reported to be endowed with a functional TLR-9 pathway (Namalwa, Raji, and HEK293-TLR9^+/+), our data suggest that transformed human cells do not sense MVMp or H-1PV either because of an absence of PRR expression or an intrinsic, or virus-driven defect in the endosomal sensing of the parvovirus genomes by TLR-9.</p> </div

Assessment of a TLR-9-dependent activation of NF-κB in HEK-BlueTM-hTLR-9 cells upon MVMp or H-1PV infection.

<p>HEK-Blue™-hTLR-9 cells (8.10⁴ cells/well) cultivated for 24 hrs in 96-well plates following the manufacturer's instructions, were infected using increasing MOIs of MVMp or H-1PV for 24 hrs. NF-κB activity was then assessed by a colorimetric assay using a reporter system measuring the release of secreted embryonic alkaline phosphatase (SEAP) in the culture medium. Control experiments consisted in the stimulation of the latter cell line, pre-treated or not for 3 hrs with the TLR-9 inhibitor ODN TTAGGG at 2 µM, with the TLR-9 agonist ODN-2395 at 4 µM. Results are expressed as means+standard deviations of three independent experiments.</p

Sensitivity to neuraminidase pre-treatment of the antiviral response triggered by parvoviruses in hPBMCs.

<p>(<b>A</b> to <b>C</b>) hPBMCs collected from the blood of healthy donors were distributed into 6-well plates at 1×10⁷ cells/5 ml culture medium/well. They were then treated or not with neuraminidase at 0.1 U/ml for 15 hrs and then mock-treated or infected with MVMp or H-1PV at 20 PFUs/cell. (<b>B</b>) In parallel to the treatment of PBMCs, HEK293 cells cultivated in 10-cm dishes at a density of 1.5 10⁶ cells/dish were also mock-treated or infected with MVMp or H-1PV at 2 PFUs/cell. Cells (<b>A</b>, <b>B</b>) as well as supernatants (<b>C</b>) were isolated 24 hrs p.i. in order to perform Southern blot (<b>A</b>), RT-PCR (<b>B</b>), and ELISA (<b>C</b>) experiments. (<b>A</b>) DNA was extracted from hPBMCs and Southern blotting performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g002" target="_blank">Figure 2</a>. (dRF, dimmer replicative form; mRF, monomeric replicative form; ssDNA, single-stranded genome). The blot shown is representative of 3 experiments which all gave similar results. (<b>B</b>) Total RNA extraction and consecutive synthesis of cDNA from hPBMC and HEK293 cultures were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g001" target="_blank">Figure 1</a>. Expression of the indicated transcripts was assessed using specific pairs of primers. Transcripts encoding the human 18S ribosomal protein were used as internal loading controls. Data shown are representative of 3 experiments that gave similar results. (<b>C</b>) Collected supernatants were centrifuged in order to discard cellular debris and analyzed by Enzyme-linked Immuno-Sorbent Assay (ELISA) for their content in IFN-α and IFN-β cytokines. Results are expressed as means+standard deviations of three independent experiments. Each presented blot is representative of 3 experiments which gave similar results.</p

Activation of both IFN-producing and IFN-signaling pathways in hPBMCs upon parvovirus infection and comparison of their intensity to that triggered by NDV.

<p>(<b>A</b>, <b>B</b> and <b>D</b>) hPBMCs collected from the blood of healthy donors were distributed into 6-well plates at 1×10⁷ cells/5 ml culture medium/well. They were then mock-treated or infected with MVMp or H-1PV at 20 PFUs/cell or with NDV (6 HU/10⁶ cells). Cultures were harvested in PBS and centrifuged at the time p.i. indicated in each figure. (<b>A</b>, <b>D</b>) One half of each pellet was resuspended in Ripa buffer supplemented with phosphatase and protease inhibitors in order to perform Western blot experiments whereas (<b>B</b>) total RNAs were extracted from the rest of each cell pellet using the RNeasy kit. (<b>A</b>, <b>D</b>) Total proteins were extracted from each sample as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#s2" target="_blank">Materials and Methods</a>. Seventy µg total proteins per sample were then subjected to 10% SDS-PAGE, transferred onto membranes, and probed with antibodies specific for total and phosphorylated STAT₂ and STAT₁ polypeptides as well as for PKR. Actin was used as an internal loading control. Each presented blot is representative of 4 additional which gave similar results. (<b>B</b>). One µg of isolated total RNA was then reverse transcribed into cDNA and 10% of this product was subjected to PCR reactions using sets of primers specific to each indicated mRNA. PCR product of 18S ribosomal RNA was used as housekeeping gene to normalize loading. No signal was detected in the samples when omitting the reverse transcriptase. Presented data are representative of 4 experiments which all gave similar results. (<b>C</b>) hPBMCs collected from the blood of healthy donors were distributed into 24-well plates at 1×10⁶ cells/500 µl culture medium/well. They were then mock-treated or infected with MVMp, H-1PV (20 PFUs/cell) or NDV (6 HU/10⁶ cells). After a period of incubation of 24 hrs media were collected, centrifuged in order to discard cellular debris and analyzed by Enzyme-linked Immuno-Sorbent Assay (ELISA) for their content in IFN-α and IFN-β. Results are expressed as means+standard deviations of seven independent experiments.</p

Defective replication of parvoviruses in hPBMCs.

<p>(<b>A</b>) hPBMCs collected from the blood of healthy donors were distributed into 6-well plates at 1×10⁷ cells/5 ml culture medium/well. They were then mock-treated or infected with MVMp or H-1PV at 10 PFUs/cell and 24 hrs later harvested for Southern blotting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g002" target="_blank">Figure 2</a>. The presented blot is representative of 3 additional which gave similar results. (<b>B</b>) hPBMCs (1×10⁷ cells/5 ml culture medium/well of a 6-well plate) and HEK293 (1.5×10⁶ cells/10-cm dish) were mock-treated or infected with the indicated parvovirus at 10 and 2 PFUs/cell respectively, and for the indicated period of time. Cultures were then harvested for Southern blotting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g002" target="_blank">Figure 2</a>. The presented blot is representative of 2 additional which gave similar results. (<b>C</b>) Expression of the parvovirus proteins in infected hPBMCs was examined in the same samples as those already analyzed for parvovirus replication in (<b>B</b>). At the time point indicated mock-treated or parvovirus-infected cultures were harvested by scraping in PBS and centrifuged. Cell pellets were then re-suspended in complete Ripa buffer supplemented with phosphatase and protease inhibitors. Total proteins were extracted from each sample as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#s2" target="_blank">Materials and Methods</a>. Fifty µg total proteins per sample were then subjected to 8% SDS-PAGE, transferred onto membranes, and probed with laboratory produced polyclonal antibodies specific for parvovirus NS1, NS2 and capsid polypeptides (Note: The antibody specific to VP proteins was designed to detect H-1PV polypeptides and is therefore less specific for MVMp). Actin was used as an internal loading control. The presented blot is representative of 2 additional which gave similar results.</p

TLR-9 dependency of type-I IFN production triggered by rodent parvoviruses in infected hPBMCs.

<p>(<b>A</b>, <b>B</b> and <b>C</b>) hPBMCs were distributed into 6-well plates at 1×10⁷ cells/5 ml culture medium/well. They were immediately pre-treated, or not, for 3 hrs with the TLR-9 inhibitor ODN TTAGGG (iODN) at 2 µM and then infected or not with MVMp or H-1PV (20 PFUs/cell). In addition, some hPBMC suspensions were directly stimulated with the TLR-9 agonist ODN 2395 at 1 µM and were used as positive control experiments. Culture supernatants (<b>A</b>, <b>B</b>) as well as total DNA (<b>C</b>) were isolated 24 hrs later to perform ELISA for type-I IFNs and Southern blot experiments, respectively, as described previously in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g001" target="_blank">Figure 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g002" target="_blank">2</a>. (<b>A</b>, <b>B</b>) Results are expressed as means+standard deviations of three independent experiments. Inhibitory effects of ODN TTAGGG on the stimulatory ODN 2395 were not assessed (ND). (<b>C</b>) (dRF, dimer replicative form; mRF, monomeric replicative form; ssDNA, single-stranded DNA genome). The blot shown is representative of 3 experiments which gave similar results.</p

Time- and MOI-dependent production and release of type-I IFNs from parvovirus-infected hPBMCs.

<p>hPBMCs collected from the blood of healthy donors were distributed into 24-well plates at 1×10⁶ cells/500 µl culture medium/well. They were then mock-treated or infected with MVMp or H-1PV at 1, 10 or 50 PFUs/cell. After a period of incubation of 15, 24 or 48 hrs media were collected, centrifuged in order to discard cellular debris and analyzed by Enzyme-linked Immuno-Sorbent Assay (ELISA) for their content in IFN-α (<b>A</b>) and IFN-β (<b>B</b>). Results are expressed as means of three experiments.</p

Activation of a TLR-9 dependent production of type-I IFNs in parvovirus-infected human Namalwa cells.

<p>The cells (1.10⁶ cells/well of a 24-well plate) were either infected with MVMp or H-1PV (∼80 PFUs/cell equivalent to 1×10⁴ virus genomes/cell), stimulated with the TLR-9 agonist ODN 2395 at 1 µM, or mock-treated. (<b>A</b>) At the indicated time points culture supernatants were collected from the respective wells and used to determine the quantity of type-I IFNs released in the culture medium by ELISA experiments. Results are expressed as means+standard deviations of three independent experiments. (<b>B</b>) Mock-treated, parvovirus-infected or ODN stimulated Namalwa cells were harvested at the indicated time points and total RNAs were extracted using the RNeasy kit as described previously in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g001" target="_blank">Figure 1</a>. Total RNAs extracted from parvovirus-infected (24 hrs p.i. at 80 PFUs/cell) or pI:C-transfected (15 hrs post-transfection with 2 µg dsRNA/ml) NB324K cells were used as positive or negative controls. Presented data are representative of 3 experiments which all gave similar results. (<b>C</b>) Total DNA was harvested at the indicated time points from parvovirus-infected (MOI of 80 PFUs/cell) or mock-treated Namalwa or HEK293T cultures to assess by Southern blot experiments as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g002" target="_blank">Figure 2</a> the replication of both parvoviruses (dRF, dimmer replicative form; mRF, monomeric replicative form; ssDNA, single-stranded DNA genome). The blot shown is representative of 3 experiments which gave similar results. (<b>D</b>) Cell pellets from mock-treated or parvovirus-infected Namalwa cultures were re-suspended in complete Ripa buffer and Western blotting was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g006" target="_blank">Figure 6</a>. Actin was used as an internal loading control. Each presented blot is representative of 3 experiments which gave similar results.</p

Time-dependent transcriptional up-regulation of type-I IFN encoding mRNAs in MVMp- or H-1PV-infected human cell lines.

<p>(<b>A</b>) HEK293 and HEK293T, (<b>B</b>) NB324K and (<b>C</b>) Hela cells were mock-treated for 24 hrs, infected with parvoviruses (5 PFUs/cell) for the indicated times, or infected with NDV (6 HU/10⁶ cells) or transfected with pI:C (2 µg/ml) for 15 hrs. At the indicated times, cells were harvested and total RNAs were extracted using the RNeasy kit. One µg was then reverse transcribed into cDNA and 10% of this product was subjected to PCR reactions using sets of primers specific to each indicated cytokine mRNA or viral transcripts. PCR product of 18S ribosomal RNA was used as housekeeping gene to normalize loading. No signal was detected in the samples when omitting the reverse transcriptase. Presented data are representative of 3 experiments which all gave similar results.</p

Quantification by real time PCR of the type-I IFN and OAS transcripts produced upon parvovirus infections of hPBMCs.

<p>hPBMCs collected from the blood of healthy donors were distributed into 24-well plates at 1×10⁶ cells/500 µl culture medium/well. They were then mock-treated or infected with MVMp, H-1PV (20 PFUs/cell) or NDV (6 HU/10⁶ cells). After a period of incubation of 24 hrs, cells were collected by centrifugation and resuspended in MagNA pure LC solution (Roche) in order to extract in an automated manner mRNAs. cDNAs were produced in the same apparatus using the first-strand cDNA synthesis kit from Roche. QRT-PCRs were then performed using specific sets of primers corresponding to the indicated transcripts. The calculated number of transcripts was normalized to the copy numbers of the housekeeping gene cyclophilin-B. Results are expressed as means+standard deviations of 6 independent experiments.</p