Increased TNF- α Initiates Cytoplasmic Vacuolization in Whole Blood Coculture with Dengue Virus

Abstract

During the acute febrile phase of dengue virus (DENV) infection, viremia can cause severe systemic immune responses accompanied by hematologic disorders. This study investigated the potential induction and mechanism of the cytopathic effects of DENV on peripheral blood cells ex vivo. At one day postinfection, there was viral nonstructural protein NS1 but no further virus replication measured in the whole blood culture. Notably, DENV exposure caused significant vacuolization in monocytic phagocytes. With a minor change in the complete blood cell count, except for a minor increase in neutrophils and a significant decrease in monocytes, the immune profiling assay identified several changes, particularly a significant reduction in CD14-positive monocytes as well as CD11c-positive dendritic cells. Abnormal production of TNF-α was highly associated with the induction of vacuolization. Manipulating TNF-α expression resulted in cytopathogenic effects. These results demonstrate the potential hematological damage caused by ex vivo DENV-induced TNF-α.

Figure 1

DENV infection of whole blood (WB) cells 24 h postincubation. (a) Experimental flowchart of this study. Following DENV (MOI = 1) infection in 100 μl of WB ex vivo for 24 h, five plasma samples were harvested to detect DENV infection. (b) The images (10x objectives) of plaque assay showed no viral replication. (c) An NS1 rapid test showed the expression of the NS1 dengue antigen in the plasma samples. Two positive controls were also performed for the plaque assay.

Figure 2

Wright-Giemsa staining images of whole blood (WB) cells 24 h postincubation. Following DENV (MOI = 1) coculture in 100 μl of WB ex vivo for 24 h, Wright-Giemsa staining, shown by an oil immersion field (100x objectives), presented histopathological changes in mononuclear cells (a). The percentages of vacuolated cells are shown (b). The quantitative data are depicted as the mean ± SD obtained from five cases. ^∗∗∗p < 0.001, compared to the mock group.

Figure 3

CBC results of whole blood (WB) cells 24 h postincubation. Following DENV (MOI = 1) coculture in 100 μl of WB ex vivo for 24 h, the CBC test showed the percentages of specific cell populations as noted. The quantitative data are depicted as the mean ± SD obtained from five cases. ^∗p < 0.05 and ^∗∗p < 0.01, compared to the mock group. ns: not significant.

Figure 4

Immune profiling in DENV-treated whole blood cells 24 h postincubation. Following DENV (MOI = 1) coculture in 100 μl of WB ex vivo for 24 h, (a) representative flow cytometric analysis and gating of various cells obtained from five cases, performed by staining for specific cell surface markers (CD4, CD8, CD11c, CD14, CD16, CD19, CD25, CD56, CD62L, and HLA-DR), in the DENV-infected and mock groups showed (b) the changes in the expression of specific immune cell populations as noted. (c) The results are shown as a percentage of the mean ± SD obtained from five cases. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001, compared to the mock group. R: region; WBC: white blood cell; bri: bright.

Figure 5

Abnormal production of TNF-α caused by DENV infection correlates with the cytopathological effects 24 h postincubation. (a) Following DENV (MOI = 1) coculture in 100 μl of WB ex vivo for 24 h, TNF-α production was measured in the plasma by ELISA. The quantitative data are depicted as the mean ± SD obtained from five cases. ^∗p < 0.05, compared to the mock group. (b) Furthermore, correlation analysis showed the strength of the relationship between TNF-α production and the induction of vacuolization, which is expressed numerically based on the r and p values.

Figure 6

Abnormal TNF-α determines the cytopathological effects 24 h postincubation. Without DENV coculture, hTNF-α (100 ng/ml) was added to the ex vivo culture of WB for 24 h. (a) Image analysis, shown by an oil immersion field (100x objectives), was performed using the Wright-Giemsa stain to show the induction of vacuolization in five cases. (b) The percentages of vacuolated cells were calculated in the counting area with a high-power field (40x objectives). The quantitative data are depicted as the mean ± SD obtained from five cases. ^∗∗∗p < 0.001, compared to the PBS group. PBS was used as a control. (c) With or without hTNF-α neutralizing antibody (Ab; 50 μg/ml) cotreatment, DENV (MOI = 1)-induced vacuolization was monitored. The percentages of vacuolated cells were calculated in three different visible areas. ^∗∗∗p < 0.001, compared to the mock group. ns: not significant.