Hemorrhage is a common clinical manifestation in dengue patients. However, the pathogenic mechanism of dengue virus (DV)-induced hemorrhage awaits clarification. We established a mouse model of DV hemorrhage using immunocompetent C57BL/6 mice by injecting DV serotype 2 strain 16681 intradermally. While inoculation of 3 ؋ 10 9 PFU of DV induced systemic hemorrhage in all of the mice by day 3 of infection, one out of three of those injected with 4 ؋ 10 7 to 8 ؋ 10 7 PFU developed hemorrhage in the subcutaneous tissues. The mice that were inoculated with 4 ؋ 10 7 to 8 ؋ 10 7 PFU but that did not develop hemorrhage were used as a basis for comparison to explore the pathogenic mechanism of dengue hemorrhage. The results showed that mice with severe thrombocytopenia manifested signs of vascular leakage and hemorrhage. We observed that high viral titer, macrophage infiltration, and tumor necrosis factor alpha (TNF-␣) production in the local tissues are three important events that lead to hemorrhage. Immunofluorescence staining revealed that DV targeted both endothelial cells and macrophages. In addition, the production of high levels of TNF-␣ in tissues correlated with endothelial cell apoptosis and hemorrhage. By comparing TNF-␣ ؊/؊ to IgH ؊/؊ , C5 ؊/؊ , and wild-type mice, we found that TNF-␣ was important for the development of hemorrhage. In vitro studies showed that mouse primary microvascular endothelial cells were susceptible to DV but that TNF-␣ enhanced DV-induced apoptosis. Our mouse model illustrated that intradermal inoculation of high titers of DV predisposes endothelial cells to be susceptible to TNF-␣-induced cell death, which leads to endothelium damage and hemorrhage development. This finding highlights the contribution of the innate immune response to dengue hemorrhage.

Activation and expansion of dengue virus-specific T cells and abnormal liver functions in dengue patients have been documented. However, it remains to be determined whether T cells are involved in the pathogenic mechanism of dengue virus infection. In this study, immunocompetent C57BL/6 mice were employed to study dengue virus-induced T cell activation. Mice were inoculated with 10(8) PFU dengue virus serotype 2 strain 16681 by the intravenous route. Dengue viral core RNA was detected by RT-PCR in mouse serum, liver, spleen, and brain at different time points after infection. Splenic T cells were activated as evidenced by their expression of CD69 and O-glycosylated CD43 at as early as day 3 after infection. Splenic T cell expression of O-glycosylated CD43 and IFN-gamma production coordinately peaked at day 5. Coincided with the peak of splenic T cell activation was hepatic lymphocyte infiltration and elevation of liver enzymes. Flow cytometric analysis revealed the infiltrating CD8(+) T cell to CD4(+) T cell ratio was 5/3. After a second inoculation of dengue virus, hepatic T cell infiltration and liver enzyme levels increased sharply. The infiltrating hepatic CD8(+) T cell to CD4(+) T cell ratio increased to 5.8/1. A strong correlation was found between T cell activation and hepatic cellular infiltration in immunocompetent mice infected with dengue virus. The kinetics of liver enzyme elevation also correlated with that of T cell activation. These data suggest a relationship between T cell infiltration and elevation of liver enzymes.

Here we summarized our findings in the mouse model of the events that lead to dengue hemorrhage. Immunocompetent mice inoculated intradermally with DENV-2 strain 16681 develop hemorrhage locally or systemically. The incidence and the severity of hemorrhage development are dependent on the size of viral inoculum. The hemorrhage mice exhibit severe thrombocytopenia, prolonged bleeding time, and increased numbers of circulating endothelial cells. In the hemorrhage tissues, there is endothelial damage accompanied by infiltrating macrophages that secret TNF-alpha. The endothelial cells express iNOS and peroxynitrite and undergo apoptosis, indicating RNS and ROS production may lead to cell death. By using mice deficient in iNOS and phox47 and apocynin, we demonstrated that RNS and ROS are important to hemorrhage development after infection by DENV. Our mouse model offers the opportunity to test potential dengue vaccines and therapeutics to treat dengue hemorrhage and to test hemorrhage induction potentials of dengue viral strains.

Vascular leakage and hemorrhagic diathesis are life-threatening complications that occur to dengue hemorrhagic patients. However, the pathogenic mechanism of hemorrhage is not well understood. It has been shown that endothelial cells are permissive to dengue virus (DV) infection in vitro and DV antigen is found in endothelial cells of autopsy specimens collected from patients who died of DHF. Therefore, the study of interaction between DV and endothelial cells will shed light on the mechanism of dengue hemorrhage. We established a dengue hemorrhage mouse model in immunocompetent C57BL/6 mice by injecting DV serotype 2 strain 16681 intradermally. While high dose DV inoculation induced hemorrhage in all of the mice, 1/3 of those injected with 4–8 x 107 PFU developed hemorrhage in the subcutaneous tissues by day 3 of infection. Importantly, hemorrhage was accompanied by severe thrombocytopenia and vascular leakage. Double immunofluorescence staining revealed that DV targeted both macrophages and endothelial cells in the subcutaneous tissues. In vitro studies showed that human umbilical cord vein endothelial cells (HUVEC) and microvascular endothelial cell line (HMEC) were susceptible to DV and TNF-α enhanced DV-induced apoptosis. In addition, DV infection induced endothelial cell NOS and ROS production and endothelial cell apoptosis, which were also enhanced by TNF-α. These findings demonstrate that dengue virus and TNF-α-induced endothelial cell apoptosis is mediated by ROS and NO.