A novel leptospiral protein increases ICAM-1 and E-selectin expression in human umbilical vein endothelial cells

It has been reported previously that activation of vascular endothelium by outer membrane proteins of the spirochetes Borrelia sp. and Treponema sp. resulted in enhanced expression of endothelial cell adhesion molecules. To investigate the role of leptospiral proteins in this process, a predicted lipoprotein encoded by the gene LIC10365 was selected, which belongs to a paralogous family that presents a domain of unknown function, DUF1565. The LIC10365 gene was cloned and the protein expressed in Escherichia coli C43 (DE3) strain using the vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and was used to assess its ability to activate cultured human umbilical vein endothelial cells. The rLIC10365 activated endothelium in such a manner that E-selectin and intercellular adhesion molecule 1 (ICAM-1) became upregulated in a dose-dependent fashion. The LIC10365-encoded protein was identified in vivo in the renal tubules of animal during experimental infection with Leptospira interrogans. Collectively, these results implicate the LIC10365-coding protein of L. interrogans as a potential effector molecule in the promotion of a host inflammatory response. This is the first report of a leptospiral protein capable of up-regulating the expression of endothelial cell adhesion molecules ICAM-1 and E-selectin.Instituto de Biotecnologia y Biologia Molecula

A novel leptospiral protein increases ICAM-1 and E-selectin expression in human umbilical vein endothelial cells

It has been reported previously that activation of vascular endothelium by outer membrane proteins of the spirochetes Borrelia sp. and Treponema sp. resulted in enhanced expression of endothelial cell adhesion molecules. To investigate the role of leptospiral proteins in this process, a predicted lipoprotein encoded by the gene LIC10365 was selected, which belongs to a paralogous family that presents a domain of unknown function, DUF1565. The LIC10365 gene was cloned and the protein expressed in Escherichia coli C43 (DE3) strain using the vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and was used to assess its ability to activate cultured human umbilical vein endothelial cells. The rLIC10365 activated endothelium in such a manner that E-selectin and intercellular adhesion molecule 1 (ICAM-1) became upregulated in a dose-dependent fashion. The LIC10365-encoded protein was identified in vivo in the renal tubules of animal during experimental infection with Leptospira interrogans. Collectively, these results implicate the LIC10365-coding protein of L. interrogans as a potential effector molecule in the promotion of a host inflammatory response. This is the first report of a leptospiral protein capable of up-regulating the expression of endothelial cell adhesion molecules ICAM-1 and E-selectin.Instituto de Biotecnologia y Biologia Molecula

A novel leptospiral protein increases ICAM-1 and E-selectin expression in human umbilical vein endothelial cells

It has been reported previously that activation of vascular endothelium by outer membrane proteins of the spirochetes Borrelia sp. and Treponema sp. resulted in enhanced expression of endothelial cell adhesion molecules. To investigate the role of leptospiral proteins in this process, a predicted lipoprotein encoded by the gene LIC10365 was selected, which belongs to a paralogous family that presents a domain of unknown function, DUF1565. The LIC10365 gene was cloned and the protein expressed in Escherichia coli C43 (DE3) strain using the vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and was used to assess its ability to activate cultured human umbilical vein endothelial cells. The rLIC10365 activated endothelium in such a manner that E-selectin and intercellular adhesion molecule 1 (ICAM-1) became upregulated in a dose-dependent fashion. The LIC10365-encoded protein was identified in vivo in the renal tubules of animal during experimental infection with Leptospira interrogans. Collectively, these results implicate the LIC10365-coding protein of L. interrogans as a potential effector molecule in the promotion of a host inflammatory response. This is the first report of a leptospiral protein capable of up-regulating the expression of endothelial cell adhesion molecules ICAM-1 and E-selectin.Instituto de Biotecnologia y Biologia Molecula

Junín Virus Infection of Human Hematopoietic Progenitors Impairs In Vitro Proplatelet Formation and Platelet Release via a Bystander Effect Involving Type I IFN Signaling

Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus (JUNV), a member of the arenaviridae family. Although a recently introduced live attenuated vaccine has proven to be effective, AHF remains a potentially lethal infection. Like in other viral hemorrhagic fevers (VHF), AHF patients present with fever and hemorrhagic complications. Although the causes of the bleeding are poorly understood, impaired hemostasis, endothelial cell dysfunction and low platelet counts have been described. Thrombocytopenia is a common feature in VHF syndromes, and it is a major sign for its diagnosis. However, the underlying pathogenic mechanism has not yet been elucidated. We hypothesized that thrombocytopenia results from a viral-triggered alteration of the megakaryo/thrombopoiesis process. Therefore, we evaluated the impact of JUNV on megakaryopoiesis using an in vitro model of human CD34+ cells stimulated with thrombopoietin. Our results showed that CD34+ cells are infected with JUNV in a restricted fashion. Infection was transferrin receptor 1 (TfR1)-dependent and the surface expression of TfR1 was higher in infected cultures, suggesting a novel arenaviral dissemination strategy in hematopoietic progenitor cells. Although proliferation, survival, and commitment in JUNV-infected cultures were normal, viral infection impaired thrombopoiesis by decreasing in vitro proplatelet formation, platelet release, and P-selectin externalization via a bystander effect. The decrease in platelet release was also TfR1-dependent, mimicked by poly(I:C), and type I interferon (IFN α/β) was implicated as a key paracrine mediator. Among the relevant molecules studied, only the transcription factor NF-E2 showed a moderate decrease in expression in megakaryocytes from either infected cultures or after type I IFN treatment. Moreover, type I IFN-treated megakaryocytes presented ultrastructural abnormalities resembling the reported thrombocytopenic NF-E2−/− mouse phenotype. Our study introduces a potential mechanism for thrombocytopenia in VHF and other diseases associated with increased bone marrow type I IFN levels

Junín virus infection of human hematopoietic progenitors impairs in vitro proplatelet formation and platelet release via a bystander effect involving type I IFN signaling

Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus (JUNV), a member of the arenaviridae family. Although a recently introduced live attenuated vaccine has proven to be effective, AHF remains a potentially lethal infection. Like in other viral hemorrhagic fevers (VHF), AHF patients present with fever and hemorrhagic complications. Although the causes of the bleeding are poorly understood, impaired hemostasis, endothelial cell dysfunction and low platelet counts have been described. Thrombocytopenia is a common feature in VHF syndromes, and it is a major sign for its diagnosis. However, the underlying pathogenic mechanism has not yet been elucidated. We hypothesized that thrombocytopenia results from a viral-triggered alteration of the megakaryo/thrombopoiesis process. Therefore, we evaluated the impact of JUNV on megakaryopoiesis using an in vitro model of human CD34+ cells stimulated with thrombopoietin. Our results showed that CD34+ cells are infected with JUNV in a restricted fashion. Infection was transferrin receptor 1 (TfR1)-dependent and the surface expression of TfR1 was higher in infected cultures, suggesting a novel arenaviral dissemination strategy in hematopoietic progenitor cells. Although proliferation, survival, and commitment in JUNV-infected cultures were normal, viral infection impaired thrombopoiesis by decreasing in vitro proplatelet formation, platelet release, and P-selectin externalization via a bystander effect. The decrease in platelet release was also TfR1-dependent, mimicked by poly(I:C), and type I interferon (IFN α/ß) was implicated as a key paracrine mediator. Among the relevant molecules studied, only the transcription factor NF-E2 showed a moderate decrease in expression in megakaryocytes from either infected cultures or after type I IFN treatment. Moreover, type I IFN-treated megakaryocytes presented ultrastructural abnormalities resembling the reported thrombocytopenic NF-E2-/- mouse phenotype. Our study introduces a potential mechanism for thrombocytopenia in VHF and other diseases associated with increased bone marrow type I IFN levels.Facultad de Ciencias Exacta

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

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

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