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Role of CD61+ cells in thrombocytopenia of dengue patients

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Abstract

Although hematological disorders with salient features of thrombocytopenia have been well documented in dengue patients, the role of CD61-expressing platelets and the megakaryocytic cell lineage in the pathogenesis of dengue virus (DENV) infection remains largely unexplored. A prospective observational study was performed using blood samples and PBMCs from dengue-confirmed patients, as well as from rhesus monkeys (RM) experimentally infected with DENV. Immunohistochemical staining and FACS techniques were applied to evaluate the frequencies of CD61+ cells that contained DENV antigen. Highly enriched population of CD61+ cells was also isolated from acute DENV-infected RM and assayed for DENV RNA by quantitative RT-PCR. Results revealed that DENV antigen was found in small vesicles of varying size, and more frequently in anucleated cells associated with platelets in dengue patients. The DENV antigen-containing cells were CD61+ and appeared to share characteristics of megakaryocytes. Kinetic profiles of CD61+ cells from DENV-infected RM revealed a transient increase in CD61+CD62P+ cells early after DENV infection. DENV RNA in a highly enriched population of CD61+ cells from the infected RM was observed during acute stage. Our results indicate that virus containing CD61+ cells may be directly linked to the platelet dysfunction and low platelet count characteristics of dengue patients.

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Acknowledgments

We would like to thank Korakot Polsrila at the Center of Excellence for Flow Cytometry and clinical staffs at the Division of Infectious Diseases, Department of Pediatrics, Faculty of Medicine Siriraj Hospital, for sample collections and laboratory confirmation of dengue. The research was supported in part by Emory SOM start-up fund, Thailand Research Fund for Senior Research Scholar, Robert E. Shop International Fellowship, the U19 Pilot Project Funds U19 AI057266 (RFA-AI-02-042), NIH/SERCEB, Emory URC grants, and the NCRR p51 support to the Yerkes National Primate Research Center DRR000165.

Conflict of interest

The authors declare no competing financial interests.

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Author notes

  1. Guey Chuen Perng

    Present address: Department of Microbiology and Immunology, College of Medicine, NCKU, Tainan City, Taiwan

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Emory Vaccine Center, Emory University School of Medicine, Dental School Building, Room 429, 1462 Clifton Road, Atlanta, GA, 30322, USA

    Sansanee Noisakran, Hui-Mien Hsiao & Guey Chuen Perng

  2. Department of Pathology and Laboratory Medicine, Emory Vaccine Center, Emory University School of Medicine, 954 Gatewood Road, Atlanta, GA, 30329, USA

    Francois Villinger

  3. Division of Pathology, Yerkes National Primate Research Center, 954 Gatewood Road, Atlanta, GA, 30329, USA

    Francois Villinger

  4. Department of Pathology and Laboratory Medicine, Emory Vaccine Center, Emory University School of Medicine, 101 Wooddruff Circle, Atlanta, GA, 30322, USA

    Aftab A. Ansari

  5. Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, 12120, Thailand

    Sansanee Noisakran

  6. Center of Excellence for Flow Cytometry, Mahidol University, Bangkok, Thailand

    Nattawat Onlamoon, Kovit Pattanapanyasat & Pucharee Songprakhon

  7. Office for Research and Development, Mahidol University, Bangkok, Thailand

    Pucharee Songprakhon

  8. Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Nasikarn Angkasekwinai

  9. Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Kulkanya Chokephaibulkit

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Electronic supplementary material

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12185_2012_1175_MOESM1_ESM.pptx

Supplementary Figure 1. A schematic diagram depicts an isolation method for megakaryocyte-like cells in circulation. Giant cells or megakaryocytic-like cells from whole blood of dengue patients was performed by applying a method developed by Wilde et al {Wilde, 1997 #2295}. Whole blood (0.3 ml) was passed down by gravity through an assembled syringe filter holder containing a nucleopore polycarbonate membrane of 5 µm pore diameter. Following two washes with saline, the membrane was removed and left to dry thoroughly before being processed for immunohistochemical staining (IHC). (PPTX 49 kb)

12185_2012_1175_MOESM2_ESM.pptx

Supplementary Figure 2. A gating strategy of flow cytometry-based cell sorting for CD41+CD61+ cells . PBMCs from dengue virus-infected rhesus monkeys on days 3 and 5 post infection were stained with a panel of fluorochromes-conjugated monoclonal antibodies with specificity for CD3, CD20, CD14, CD16, CD41 and CD61 and processed for a FACSAria II cell sorter. CD3-CD20-CD14-CD16- cells were selected from the total stained cell population and those with CD41+CD61+, CD41-CD61+ and CD41-CD61- subsets were collected in each sample for further investigation. Pre-sorted and post-sorted cell populations were analyzed for the purity of cell sorting. (PPTX 211 kb)

12185_2012_1175_MOESM3_ESM.pptx

Supplementary Figure 3. Detection of dengue viral antigen in CD61+ cells. Blood smears prepared from dengue patients were subjected to double immunofluorescence staining for dengue viral antigen and CD61 as described in the Methods section. The stained cells were mounted with DAPI and observed using a Zeiss fluorescence microscope equipped with an Axis 5 digital camera. Dengue viral antigen was observed in granular cells with CD61 marker. Results show bright field, fluorescent field and merged images from blood smears of 2 randomly selected dengue patients. Dengue viral antigen (red); CD61 (green); nucleus (blue); co-localization of dengue viral antigen with CD61 (yellow). (PPTX 382 kb)

12185_2012_1175_MOESM4_ESM.pptx

Supplementary Figure 4. The presence of giant platelets and DENV antigen-positive platelets in dengue patients. Whole blood from dengue patients was smeared onto slides and subjected to Wright’s stain. The stained cells were observed using a light microscope with 40× (A) and 100× (B) magnification of objective lenses. Results revealed the presence of giant platelets with budding vesicles (red arrows) in the samples from dengue patients. To detect dengue viral antigens in the platelets, double immunohistochemical staining was performed using platelets isolated from whole blood of dengue patients. A small proportion of the platelets were found to contain dengue viral antigen as indicated by red arrows (C and D). Representative images from two dengue patients are shown. (PPTX 772 kb)

12185_2012_1175_MOESM5_ESM.pptx

Supplementary Figure 5. Dengue viral antigen was observed in anucleated cells and irregular shaped-nucleus containing cells with loose cytoplasm. Immunohistochemical staining for dengue viral antigen was performed on PBMC smears from dengue patients as described in the Materials and Methods. Mouse anti-dengue E antibody (A and C) and isotype-matched control antibody (B and D) were used in the primary staining step. Counterstaining with hematoxylin was applied to all the stained samples. Representative images taken from different areas on the slides are shown (dengue viral antigen, brown). (PPTX 802 kb)

12185_2012_1175_MOESM6_ESM.pptx

Supplementary Figure 6. CD41+ cells with budding platelets and proplatelet formation contained dengue viral antigen. PBMC smears prepared from dengue patients were processed for double immunohistochemical staining for dengue viral antigen (red) and CD41(dark blue), a marker for platelets and megakaryocytes, as described in the Materials and Methods. Mouse anti-dengue E antibody (A and B) and isotype-matched control antibody (C) were utilized in the primary staining step. The stained cells were mounted with Hoechst and observed using a fluorescence microscope. Results show bright field, fluorescent field and merged image of the stained cells captured from different areas on the slides. (PPTX 367 kb)

12185_2012_1175_MOESM7_ESM.pptx

Supplementary Figure 7. Dengue viral antigen was observed in CD41+ cells with low cytoplasm to nucleus ratio in association with platelets. Double immunohistochemical staining was performed to determine dengue viral antigen (red) and CD41(dark blue), a marker for platelets and megakaryocytes as described in the Materials and Methods using PBMC smears from dengue patients. A and B show two representative images captured from different areas on the stained slides. (PPTX 565 kb)

12185_2012_1175_MOESM8_ESM.pptx

Supplementary Figure 8. A gating strategy of multicolor flow cytometric analysis for CD61+CD62P+ cells in leukocyte subpopulation. Whole blood from dengue virus-infected rhesus monkeys were stained with a panel of fluorochromes-conjugated monoclonal antibodies with specificity for CD45, CD3, CD20, CD14, CD61 and CD62P. The stained cells were analyzed by flow cytometry for the frequency of CD61+CD62+ cells in the following leukocyte subpopulations: granulocytes (CD45+CD14-CD3-CD20-), monocytes (CD45+CD14+CD3-CD20-), T lymphocytes (CD45+CD14-CD3+CD20-), B lymphocytes (CD45+CD14-CD3-CD20+) and non-T, non-B lymphocytes (CD45+CD14-CD3-CD20-). (PPTX 431 kb)

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Noisakran, S., Onlamoon, N., Pattanapanyasat, K. et al. Role of CD61+ cells in thrombocytopenia of dengue patients. Int J Hematol 96, 600–610 (2012). https://doi.org/10.1007/s12185-012-1175-x

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