1. FREQUENCIES AND PHENOTYPIC CHANGES OF DENDRITIC CELLS DURING ACUTE DENGUE VIRAL INFECTION
Lertjuthaporn S1, Khowawisetsut L2, Keawvichit R1, Thitilertdecha P3, Onlamoon N3, Pattanapanyasat K3
1Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University,
2Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University,
3Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University

2. Objectives Materials & Methods
Dengue virus (DENV) is the most prevalent arthropod-borne viral disease in humans. DENV causes a spectrum of illness ranging from mild to potentially severe complications. There is no licensed vaccines or effective antiviral drugs available at present. Dendritic cells (DCs) play a crucial role in initiating and regulating a potent antiviral immune response. This study was conducted to comparatively characterize the frequencies and phenotypic changes of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in DENV-infected patients with dengue fever (DF), dengue hemorrhagic fever (DHF) and healthy controls.
Twenty-two DENV-infected patients from Ramathibodi Hospital and Siriraj Hospital and fifteen healthy returned subjects were recruited into this study. Sixty-eight blood samples from DENV-infected patients were collected in different days of fever. DENV cases were confirmed by a serotype-specific reverse transcription-PCR (RT-PCR) and defined by their severity as dengue fever (DF) or dengue hemorrhagic fever (DHF) according to the WHO 1997 guidelines. Two major subsets of human blood DCs, mDCs (Lin-CD11c+CD123-) and pDCs (Lin-CD11c-CD123+) were analyzed by polychromatic flow cytometry.
Dengue classification (adapted from WHO 1997 guidelines)
Surface staining with antibodies
CD3-FITC
HLA-DR-PE
CD19-PE-TxRd
CD45-PerCP
CD69-PE-Cy7
CD7-APC
CD11C-A700
CD56-APC-Cy7
CD16-BV510
CCR3-BV605
CD14-BV570
CD123-BV650
Symptomatic
Dengue hemorrhagic fever
(DHF)
Undifferentiated fever
(viral syndrome)
Dengue virus infection
Dengue fever
(DF)
Asymptomatic
Whole blood
BD LSRFortessa flow cytometer

3. Results Identification of DC subsets in peripheral blood
Fig.1. Gating strategy for analysis of DCs from peripheral blood. Two major populations of DC subsets were identified from CD45+/CD14-/CD3-/CD19-/CD7-/HLA-DR+ cells. mDCs were identified as Lin-CD11C+CD123-, while pDCs were identified as Lin-CD11C-CD123+. Double negative (DN) subset (CD45+/CD14-/CD3-/CD19-/CD7-/HLA-DR+/CD11c-CD123-) was also observed in DENV-infected patients.
FSC-H
SSC-W
FSC-W
SSC-A
FSC-A
SSC-H
FSC-H
PerCP: CD45
PE-TxRed: CD19
SSC-A
SSC-A
SSC-A
BV510: CD16
BV570: CD14
FSC-A
FITC: CD3
mDC subset
APC: CD7
pDC subset
A700: CD11C
Double negative (DN) subset
PE: HLA-DR
BV650: CD123

4. Results
Distribution of DC subsets in DENV-infected patients and healthy subjects A B C
Fig.2. The distribution of DC subsets were analyzed in blood samples from 68 DENV-infected samples and 14 healthy samples. The average percentage of mDCs, pDCs and double negative (DN) cells from peripheral blood of healthy subjects were 81.39%, 13.19% and 5.42%, respectively. The average percentage of mDCs in DENV-infected patients was significantly decreased when compared with that of healthy individuals (39.19% versus 81.39%, respectively, p  ). In contrast, the average percentage of DN subset in DENV-infected patients was significantly higher than that of healthy individuals (38.77% versus 5.42%, p  ). There is no significant difference between healthy individuals and DENV-infected patients in the frequencies of pDC subset.

5. The frequencies of DC subsets between DF and DHF patients
Results
The frequencies of DC subsets between DF and DHF patients A B C
Fig.3. The percentages of DC subsets were compared between DF and DHF patients. Thirty-one of the patients had DF and thirty-seven of the remaining had DHF. The percentages of mDC subset in DHF patients was significantly lower than DF patients (*** p ≤ , by Mann-Whitney U test). In contrast, the percentages of DN subset in DHF patients was significantly higher than DF patients (* p ≤ 0.05 by Mann-Whitney U test). There is no significant difference between DF and DHF patients in the frequencies of pDC subset.

6. Kinetic change of the DCs frequencies during acute DENV infection
Results
Kinetic change of the DCs frequencies during acute DENV infection A B C
--- Healthy subjects
⎯⎯ DENV-infected patients
Phase of DENV
Febrile phase = D-2 to D-1
Defervescense phase = D0 to D+1
Afebrile phase = D+2
Fig.4. mDCs, pDCs and DN cells were compared in relation to day of defervescence (D0). The percentages of mDCs from DENV-infected patients were significantly decreased from day -2 (febrile phase) to day +2 (afebrile phase). The percentages of pDCs from DENV-infected patients were significantly increased from day -2 (febrile phase) and then decreased from day 0 (defervescense phase) to a normal level at day +2 (afebrile phase). In contrast, the percentage of DN from DENV-infected patients were significantly increased from day-2 (fever phase) to day+2 (afebrile phase).

7. Conclusions Acknowledgements
“Frequencies and Phenotypic Changes of Dendritic Cells during Acute Dengue Viral Infection”
Our results showed a number of differences among the DC subsets, DENV-infected patients had lower percentages of mDCs when compared to healthy subjects and that the percentages were significantly decreased from febrile phase to afebrile phase. In contrast, the patients had higher percentages of DN subset and were significantly increased from febrile phase to afebrile phase. Although there was no difference in the percentage of pDCs between the patients and the healthy subjects, the percentages of pDCs from the patients were significantly increased from febrile phase and then decreased to a normal level at the afebrile phase. This study provided key insight into the contribution of the difference of DC phenotypes and their kinetics during acute DENV infection.
Acknowledgements
This study was financial supported by the NIH grant no. 1R01AI and the Royal Golden Jubilee (RGJ)-Ph.D. Program (PHD/0111/2554) under the Thailand Research Fund (TRF).