The patients’ sera data were expressed as the median± interquartile range and tested if the val-ues come from a Gaussian distribution by using D’Agostino and Pearson omnibus normality test. If the data meet Gaussian distribution, the significance of differences between each groups was analyzied using One-way ANOVA with Tukey’s method. If the data do not meet the assumptions of normality, they were analyzed with a non-parametric test by Kruskal-Wallis test. Thein vitro and in vivo data are expressed as the mean ± standard deviation (SD) from more than three independent experiments. Student’s t-test was used to analyze the significance
of differences between the test and control groups. One-way ANOVA with Kruskal-Wallis comparison test was used to analyze the significance of differences between multiple groups.
All data were analyzed by GraphPad Prism 5 software. P values <0.05 were considered statisti-cally significant.
Supporting information
S1 Table. Characteristics of dengue patients.
(DOCX)
S1 Fig. The correlations of serum NS1, HPA-1, MMP-9, CD138, MIF levels and viral load in severe dengue patients. The correlations of the concentrations of (A) NS1, (B) HPA-1, (C) MMP-9, (D) CD138 and (E) MIF and viral load in the same group of severe dengue patients were plotted. Linear regressions were analyzed using nonparametric correlation test (panel A, B, C, D and E).
(DOCX)
S2 Fig. DENV NS1 induces HPA-1 activation and vascular leakage. (A) HUVECs were treated with PBS, NS1 or NS1 mixed with anti-NS1 antibodies (2E8) or control mouse IgG (cmIgG) for 24 h, and the HPA-1 level was determined by western blot. The relative HPA-1 protein level (including the proform and active form) was normalized toβ-actin, and the fold change is noted under each band. (B) BALB/c mice were intravenously injected with Evans Blue dye, followed by subcutaneous injections of PBS or different doses of HPA-1, heat-dena-tured HPA-1 or thrombin. After the mice were sacrificed, skin samples were collected and pro-cessed 3 h postinjection.
(DOCX)
S3 Fig. DENV NS1-induced MIF secretion causes glycocalyx degradation and hyperperme-ability in HUVECs. (A) HUVECs were treated with different concentrations of NS1 for the indicated times, followed supernatant collection for the detection of MIF by ELISA. (B) HUVEC monolayers were incubated with the supernatant from control or NS1-treated HUVEC cultures for the indicated times, and the endothelial permeability was then deter-mined by Transwell permeability assay. (C) HUVEC monolayers were incubated with control or NS1-treated HUVEC-conditioned medium for 24 h. The concentration of CD138 in the supernatant after the incubation was determined by ELISA. (D) (E) Control or NS1-treated HUVEC culture supernatant with or without anti-MIF polyclonal antibodies or HPA-1 inhibi-tor (OGT 2115) or anti-NS1 antibodies (2E8) and incubated with HUVECs for 24 h. (D) The endothelial permeability was determined by Transwell permeability assay, and (E) the concen-tration of CD138 in the supernatant was determined by ELISA. (F) HUVEC monolayers were treated with PBS, NS1 or NS1 mixed with anti-NS1 antibodies (2E8) or HPA-1 inhibitor (OGT 2115) for the indicated times, and the concentration of MIF in the supernatant was determined by ELISA; S/N, supernatant;P<0.05,P<0.005,P<0.001; unpaired t-test (panel B and C), Kruskal-Wallis ANOVA (panel D and E).
(DOCX)
S4 Fig. MIF induces HPA-1 activation and glycocalyx shedding in HUVECs. (A) HUVECs were treated with or without MIF recombinant protein (1μg/ml) for the indicated times, and the concentration of CD138 in the supernatant was determined by ELISA. (B) HUVECs were treated with or without MIF recombinant protein (1μg/ml) for 18 h, and the HPA-1 level was determined by western blot. The relative HPA-1 protein level (including the proform and active form) was normalized toβ-actin, and the fold change is noted under each band. (C)
HUVECs were treated as indicated for 18 h and then stained for HPA-1 (red), CD138 (green), and nuclei (blue).P<0.05,P<0.005; unpaired t-test (panel A).
(DOCX)
S5 Fig. DENV NS1 does not induce MIF and MMP-9 secretion in PBMCs. (A) (B) Isolated human PBMCs were treated with or without NS1 (10μg/ml) for the indicated times, and the concentration of (A) MIF and (B) MMP-9 in the supernatant was determined by ELISA.
(DOCX)
S6 Fig. DENV NS1-induced MMP-9 secretion from WBCs causes endothelial hyperperme-ability. Isolated human WBCs were treated with or without NS1 for 24 h, and the supernatants were collected. HUVEC monolayers were incubated with the supernatant from control or NS1-treated WBCs for 6 h; then, endothelial permeability was determined by Transwell per-meability assay. S/N, supernatant;P<0.05; Kruskal-Wallis ANOVA.
(DOCX)
S7 Fig. Cytokine secretion profile of WBCs and THP-1 cells after DENV NS1 stimulation.
(A) (B) (C) Isolated human WBCs and (D) (E) (F) THP-1 cells were treated with or without NS1 for the indicated times, and the concentration of MIF, IL-6 and IL-8 in the supernatant was determined by ELISA;P<0.05,P<0.005,P<0.001; unpaired t-test (panel A, B, C and D).
(DOCX)
S8 Fig. DENV NS1-induced endothelial hyperpermeability is mediated by MIF. (A) HUVECs were transfected with MIF shRNA (shMIF) or scrambled shRNA (shLuc). The cell lysates were collected, and the relative protein level of MIF was measured by western blot. (B) The permeability of shMIF HUVECs and shLuc HUVECs after 24 h of NS1 treatment was detected by Transwell permeability assay. The results are presented as the mean± SD of tripli-cate measurements.
(DOCX)
S9 Fig. Inhibition of MIF and MMP-9 attenuate NS1-induced vascular leakage in mice.
BALB/c mice were intravenously injected with Evans Blue dye, followed by the subcutaneous injection of PBS or different doses of NS1, NS1 with MMP-9 inhibitor I or NS1 with ISO-1 for 6 h. After 5 h, the mice were subcutaneously injected with thrombin as a positive control. After another hour, the mice were sacrificed, and skin samples were collected and processed.
(DOCX)
S10 Fig. The correlations of serum levels of HPA-1 with NS1 and MIF in severe dengue patients. (A) The correlations of the concentrations of (A) NS1, (B) MIF, and HPA-1 in the severe dengue patients were plotted. Linear regressions were analyzed using nonparametric correlation test (panel A and B).
(DOCX)
Acknowledgments
We would like to thank the members of the Center of Infectious Disease and Signaling Research of NCKU for their invaluable input and insight throughout the course of this study. We also thank the technical services provided by the “Bioimaging Core Facility of the National Core Facility for Biopharmaceuticals, Ministry of Science and Technology, Taiwan”.
Author Contributions
Conceptualization: Hong-Ru Chen, Trai-Ming Yeh.
Investigation: Hong-Ru Chen, Chiao-Hsuan Chao.
Methodology: Hong-Ru Chen.
Resources: Ching-Chuan Liu, Tzong-Shiann Ho, Huey-Pin Tsai, Guey-Chuen Perng, Jen-Ren Wang.
Supervision: Trai-Ming Yeh.
Writing – original draft: Hong-Ru Chen.
Writing – review & editing: Guey-Chuen Perng, Yee-Shin Lin, Trai-Ming Yeh.
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日期：107 年 4 月 2 日
一、 參加會議經過
2018 年亞太生命科學及生物工程會議於 3/27-3/29 在日本京都舉行，我於 3/26 早上搭長榮 航空飛機由高雄出發，中午抵達大阪關西機場，入關後轉日本 JR 火車下午抵達京都，3/27 到會場報到，3/28-29 兩天早上聆聽大會安排的特別演講，下午海報展示。這次大會因為在 日本地理位置和台灣較近，而且同一場地還有資訊工程方面的會議在舉行，所以有有許多 台灣各個學校來自不同領域的學者和學生參加，大家也很難得的可以在這幾天跨領域的交 流互動。