Figure 1.
Characterization of liquid cultures of human CD34+ cells stimulated by TPO.
CD34+ cells (1×104/ml) purified by immunomagnetic positive selection were cultured in IMDM containing 5% human serum and TPO (25 ng/ml added at days one and seven of the cell culture). At the indicated culture times, (A) total cell count was determined with a hemocytometer and (B) cell size was analyzed by flow cytometry. (C) CD41 and CD34 expression were evaluated by labeling cells with specific mAbs or corresponding matched isotypes and establishing the percentage of positive cells by flow cytometry analysis. (D) Platelet (Plt) count was evaluated by flow cytometry and cellular apoptosis was determined by detecting nuclear morphological changes of cells stained with acridine orange and ethidium bromide by fluorescence microscopy. (E) Culture morphology was assessed by phase contrast microscopy [original magnification 450×, except day 12 inset (1200×)]. The values expressed in panels A–D represent the mean ± SEM of five independent experiments. Panel E shows a representative experiment of five similar replicates.
Figure 2.
Influence of JUNV infection on cellular apoptosis, proliferation, clonogenic capacity and megakaryocyte development of TPO-stimulated CD34+ cells.
CD34+ cells were infected with JUNV at a MOI of one or the equivalent volume of UV-irradiated virus or Vero cell supernatant (mock) for one hr at 37°C, washed, and then stimulated with TPO. (A) Apoptosis, (B) total cell count, (C) megakaryocyte colonies grown in collagen-based serum-free medium containing 50 ng/ml TPO, percentages of (D) CD41+ and (E) CD42b+ cells and (F) ploidy distribution were determined at the indicated days of culture, except for colonies and ploidy, which were counted after 12 or 14 days of culture, respectively. The values represent the mean ± SEM of four independent experiments.
Figure 3.
JUNV infection impairs proplatelet production and platelet release.
CD34+ cells were mock-infected or infected with UV-irradiated JUNV or JUNV and then stimulated with TPO. (A) The number of megakaryocytes displaying proplatelets was determined at day 12 of the culture by F-actin and nuclear staining with phalloidin-TRITC and DAPI, respectively, and examined by fluorescence microscopy. The values represent the mean ± SEM of four independent experiments. (B) A representative picture of a proplatelet-displaying megakaryocyte. (C) Plt count was determined by flow cytometry. At different days, culture aliquots were stained with an anti-CD41-FITC mAb and the acquisition rate was fixed at 1 µl/sec for 100 sec. Events were collected without gating using a log scale for size (FSC) and intracellular granularity (SSC). An analytical gate was determined based on the scatter properties of peripheral blood platelets treated similarly. Culture-derived platelets were counted as CD41+ events with the same scatter properties as blood platelets. The values are expressed as platelets (×105) and represent the mean ± SEM of six independent experiments. * indicates p<0.05 vs. UV-irradiated JUNV or mock. (D) A representative flow cytometric analysis 15 days after TPO stimulation.
Figure 4.
JUNV replication in CD34+ cells.
CD34+ cells were inoculated with UV-irradiated JUNV or JUNV and stimulated with TPO for ten days. Viral replication was assayed by RT-PCR, immunofluorescence and flow cytometry. (A) RT-PCR studies. (B) To detect JUNV antigens by immunofluorescence cells were washed, cytocentrifuged on silanized glasses, fixed, permeabilized and incubated first with a pool of specific mAbs against JUNV and a rabbit-anti-human vWF polyclonal Ab to identify megakaryocytes, and then with FITC-conjugated anti-rabbit (green) and Cy3-conjugated anti-mouse Igs (red). The slides were counterstained with DAPI and photographed at 1000× magnification. (C) Cells were stained as in B and then analyzed by flow cytometry. (D) As a positive control, JUNV-susceptible Vero-76 cells were inoculated with JUNV and seven days later were stained with the pool of specific mAbs against JUNV followed by Cy3-anti-mouse Igs. Negative controls in B and C were performed by incubating cells only with secondary Abs. Panels show a representative experiment of three similar replicates.
Figure 5.
Role of TfR1 in JUNV infection and impaired platelet production.
(A) The kinetics of TfR1 expression after JUNV infection in CD34+ cells stimulated with TPO were determined by flow cytometry. (B) Receptor expression was detected in UV-irradiated JUNV- and JUNV-infected cells incubated with FITC-anti-CD71 (anti-TfR1) mAb or with a matched isotype control. The histogram depicts a representative flow cytometric analysis of TfR1 staining after 120 hr of infection. (C) CD34+ cells were pre-incubated with an anti-CD71, anti-HLA-ABC mAb or ferric ammonium citrate (FAC, 10 µg/ml) for 1 hr (to down-regulate TfR1). Cells were then infected with JUNV and stimulated with TPO and viral antigens were detected by flow cytometry. The figure shows a representative experiment of three similar replicates. (D) CD34+ cells were treated as mentioned in C, and also with deferoxamine (1 µM) for 24 hr (to up-regulate TfR1). Platelets produced in culture were counted at day 15. The values represent the mean ± SEM of three independent experiments,* indicates p<0.05 vs. UV-irradiated JUNV, # indicates p<0.05 vs. JUNV.
Figure 6.
The role of the IFN β pathway in platelet production and JUNV infection.
(A) CD34+ cells were treated with poly(I:C) (100 µg/ml) at the indicated days, stimulated with TPO, and Plt counts were determined at day 15 of the culture. The values represent the mean ± SEM of four independent experiments, * p<0.05 vs. vehicle (control). (B) IFN β mRNA levels in UV-irradiated JUNV- or JUNV-infected CD34+ cells were determined by RT-PCR at the indicated days of culture. The figure shows a representative experiment of three similar replicates. (C) CD34+ cells were treated with IFN β and stimulated with TPO. Plt counts were determined at day 15 of the culture. The values represent the mean ± SEM of three independent experiments,* indicates p<0.05 vs. no IFN β, # indicates p<0.05 vs. IFN β (10 U/ml). (D) Total cell number was determined at the indicated days of culture by counting cells with a hemocytometer. Similar results were obtained in MTT assays. The values represent the mean ± SEM of three independent experiments. (E) Anti-IFN β (1,000 neutralizing units) or an equal volume of rabbit Igs was added before CD34+ cell infection and Plt counts were determined at day 15. Values represent the mean ± SEM of three independent experiments, * indicates p<0.05 vs. UV-irradiated JUNV. (F) Type I IFN receptor subunit (IFNAR1 and 2) mRNAs were evaluated by RT-PCR in megakaryocyte precursors purified by immunomagnetic positive selection (99±1% of purity). The figure shows a representative experiment of three similar replicates. (G) CD34+ cells were infected with JUNV and 1000 neutralizing units of anti-IFN β or an equal volume of rabbit Igs were added before TPO stimulation. Viral antigens were detected by flow cytometry. The figure shows a representative experiment of two similar replicates. (H) The kinetics of TfR1 expression in the presence or absence (vehicle) of IFN β (10 U/ml) in CD34+ cells stimulated with TPO were determined by flow cytometry.
Figure 7.
Intracellular mechanisms involved in the JUNV-induced inhibition of platelet production.
CD34+ cells were UV-irradiated JUNV- or JUNV-infected, washed and stimulated with TPO. (A) The Src inhibitor PP2 (10 µM) was added at the indicated days, and Plt counts were determined at culture day 15. The values represent the mean ± SEM of four independent experiments, * indicates p<0.05 vs. UV-irradiated JUNV-treated cells. (B) Semi-quantitative RT-PCR analysis of relevant molecules involved in megakaryo/thrombopoiesis were performed at the indicated days of culture. The figure shows a representative experiment of three similar replicates. (C) NF-E2 expression was assessed in the megakaryocytic population by immunostaining the cells first with a PE-conjugated anti-CD41 mAb or an isotype-matched control. Then the cells were incubated with anti-NF-E2 polyclonal followed by FITC-conjugated swine anti-rabbit Igs. Cells were analyzed by flow cytometry. Non-specific fluorescence was assessed using rabbit serum instead of primary Ab. The values represent the mean ± SEM of three independent experiments,* indicates p<0.05 vs. UV-irradiated JUNV-infected cells. The histogram shows a representative flow cytometric analysis at day ten.
Figure 8.
NF-E2 expression and ultrastructural studies of megakaryocytes treated with IFN β.
(A) After 12 days of CD34+ cell TPO stimulation, megakaryocytes were purified by immunomagnetic positive selection (98±1% of purity) and NF-E2 expression was determined two or four days after IFN β (10 U/ml) treatment using the anti-NF-E2 polyclonal Ab (or rabbit serum) followed by FITC-conjugated swine anti-rabbit Igs. The values represent the mean ± SEM of three independent experiments, * indicates p<0.05 vs. vehicle. (B) Ultrastructure of megakaryocytes cultured in the presence of vehicle or IFN β from day seven to day fourteen. Inset in the upper panel shows culture-derived platelets observed only in vehicle-treated samples.
Figure 9.
Model of JUNV infection of CD34+ cells stimulated with TPO.
JUNV induces an increase in the expression of TfR1. A very low proportion of megakaryocytes are infected by JUNV, but the infection triggers type I IFN which impairs proplatelet production, platelet release and platelet function. The concomitant down-regulation of NF-E2 in the megakaryocytic population may be involved in this effect.
Table 1.
Primers used for RT-PCR (5′→3′).