Video_2_Platelets Promote Brucella abortus Monocyte Invasion by Establishing Complexes With Monocytes.AVI

<p>Brucellosis is an infectious disease elicited by bacteria of the genus Brucella. Platelets have been extensively described as mediators of hemostasis and responsible for maintaining vascular integrity. Nevertheless, they have been recently involved in the modulation of innate and adaptive immune responses. Although many interactions have been described between Brucella abortus and monocytes/macrophages, the role of platelets during monocyte/macrophage infection by these bacteria remained unknown. The aim of this study was to investigate the role of platelets in the immune response against B. abortus. We first focused on the possible interactions between B. abortus and platelets. Bacteria were able to directly interact with platelets. Moreover, this interaction triggered platelet activation, measured as fibrinogen binding and P-selectin expression. We further investigated whether platelets were involved in Brucella-mediated monocyte/macrophage early infection. The presence of platelets promoted the invasion of monocytes/macrophages by B. abortus. Moreover, platelets established complexes with infected monocytes/macrophages as a result of a carrier function elicited by platelets. We also evaluated the ability of platelets to modulate functional aspects of monocytes in the context of the infection. The presence of platelets during monocyte infection enhanced IL-1β, TNF-α, IL-8, and MCP-1 secretion while it inhibited the secretion of IL-10. At the same time, platelets increased the expression of CD54 (ICAM-1) and CD40. Furthermore, we showed that soluble factors released by B. abortus-activated platelets, such as soluble CD40L, platelet factor 4, platelet-activating factor, and thromboxane A₂, were involved in CD54 induction. Overall, our results indicate that platelets can directly sense and react to B. abortus presence and modulate B. abortus-mediated infection of monocytes/macrophages increasing their pro-inflammatory capacity, which could promote the resolution of the infection.</p

Role of TfR1 in JUNV infection and impaired platelet production.

<p>(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.</p

NF-E2 expression and ultrastructural studies of megakaryocytes treated with IFN β.

<p>(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.</p

Intracellular mechanisms involved in the JUNV-induced inhibition of platelet production.

<p>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.</p

Image_1_Platelets Promote Brucella abortus Monocyte Invasion by Establishing Complexes With Monocytes.TIF

<p>Brucellosis is an infectious disease elicited by bacteria of the genus Brucella. Platelets have been extensively described as mediators of hemostasis and responsible for maintaining vascular integrity. Nevertheless, they have been recently involved in the modulation of innate and adaptive immune responses. Although many interactions have been described between Brucella abortus and monocytes/macrophages, the role of platelets during monocyte/macrophage infection by these bacteria remained unknown. The aim of this study was to investigate the role of platelets in the immune response against B. abortus. We first focused on the possible interactions between B. abortus and platelets. Bacteria were able to directly interact with platelets. Moreover, this interaction triggered platelet activation, measured as fibrinogen binding and P-selectin expression. We further investigated whether platelets were involved in Brucella-mediated monocyte/macrophage early infection. The presence of platelets promoted the invasion of monocytes/macrophages by B. abortus. Moreover, platelets established complexes with infected monocytes/macrophages as a result of a carrier function elicited by platelets. We also evaluated the ability of platelets to modulate functional aspects of monocytes in the context of the infection. The presence of platelets during monocyte infection enhanced IL-1β, TNF-α, IL-8, and MCP-1 secretion while it inhibited the secretion of IL-10. At the same time, platelets increased the expression of CD54 (ICAM-1) and CD40. Furthermore, we showed that soluble factors released by B. abortus-activated platelets, such as soluble CD40L, platelet factor 4, platelet-activating factor, and thromboxane A₂, were involved in CD54 induction. Overall, our results indicate that platelets can directly sense and react to B. abortus presence and modulate B. abortus-mediated infection of monocytes/macrophages increasing their pro-inflammatory capacity, which could promote the resolution of the infection.</p

JUNV replication in CD34⁺ cells.

<p>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.</p

Characterization of liquid cultures of human CD34⁺ cells stimulated by TPO.

<p>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.</p

The role of the IFN β pathway in platelet production and JUNV infection.

<p>(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.</p

Influence of JUNV infection on cellular apoptosis, proliferation, clonogenic capacity and megakaryocyte development of TPO-stimulated CD34⁺ cells.

<p>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.</p