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.

The clinical picture of severe acute respiratory syndrome (SARS) is characterized by pulmonary inflammation and respiratory failure, resembling that of acute respiratory distress syndrome. However, the events that lead to the recruitment of leukocytes are poorly understood. To study the cellular response in the acute phase of SARS coronavirus (SARS-CoV)-host cell interaction, we investigated the induction of chemokines, adhesion molecules, and DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin) by SARS-CoV. Immunohistochemistry revealed neutrophil, macrophage, and CD8 T-cell infiltration in the lung autopsy of a SARS patient who died during the acute phase of illness. Additionally, pneumocytes and macrophages in the patient's lung expressed P-selectin and DC-SIGN. In in vitro study, we showed that the A549 and THP-1 cell lines were susceptible to SARS-CoV. monocytes, and activated T cells in a chemotaxis assay. We also demonstrated that DC-SIGN was inducible in THP-1 as well as A549 cells after SARS-CoV infection. Our in vitro experiments modeling infection in humans together with the study of a lung biopsy of a patient who died during the early phase of infection demonstrated that SARS-CoV, through a dynamic interaction with lung epithelial cells and monocytic cells, creates an environment conducive for immune cell migration and accumulation that eventually leads to lung injury.Severe acute respiratory syndrome (SARS) in adults causes new pulmonary infiltration, lymphopenia, thrombocytopenia, and high levels of proinflammatory cytokines and chemokines (30) and C-reactive protein (28) in the sera. The clinical picture is characterized by a cascade of immunological events leading to pulmonary inflammation and respiratory failure (9, 17), resembling adult acute respiratory distress syndrome (ARDS) (8). High levels of chemokines and cytokines, triggered by the host immune response to SARS coronavirus (SARS-CoV), are believed to contribute to the progressive pulmonary infiltration of macrophages (16), polymorphonuclear leukocytes (2), and T cells (11) and to eventual diffuse alveolar damage and fibrosis (12). However, it remains to be determined how the cellular response in the early stage of virus-host cell interaction results in the sequence of events that leads to the severe clinical outcome.In situ hybridization and immunohistochemistry revealed that both SARS-CoV nucleic acids and antigens are present within type II pneumocytes (26). Alveolar macrophages are also reported to harbor SARS-CoV (23). Hence, it is important to investigate how the interaction between SARS-CoV and pneumocytes and macrophages influences the subsequent events in the lung.DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin) is a type II C-type lectin that is naturally expressed in human dendritic cells. It has been reported that DC-SIGN binds SARS-CoV and mediates its entry into myeloid dendritic cells by binding to the spike protein (31). However, the inducibility of DC-SIGN in cells encountering the virus and its si...

We examined the extent to which CXCR3 mediates resistance to dengue infection. Following intracerebral infection with dengue virus, CXCR3-deficient (CXCR3−/−) mice showed significantly higher mortality rates than wild-type (WT) mice; moreover, surviving CXCR3−/− mice, but not WT mice, often developed severe hind-limb paralysis. The brains of CXCR3−/− mice showed higher viral loads than those of WT mice, and quantitative analysis using real-time PCR, flow cytometry, and immunohistochemistry revealed fewer T cells, CD8+ T cells in particular, in the brains of CXCR3−/− mice. This suggests that recruitment of effector T cells to sites of dengue infection was diminished in CXCR3−/− mice, which impaired elimination of the virus from the brain and thus increased the likelihood of paralysis and/or death. These results indicate that CXCR3 plays a protective rather than an immunopathological role in dengue virus infection. In studies to identify critical CXCR3 ligands, CXCL10/IFN-inducible protein 10-deficient (CXCL10/IP-10−/−) mice infected with dengue virus showed a higher mortality rate than that of the CXCR3−/− mice. Although CXCL10/IP-10, CXCL9/monokine induced by IFN-γ, and CXCL11/IFN-inducible T cell α chemoattractant share a single receptor and all three of these chemokines are induced by dengue virus infection, the latter two could not compensate for the absence of CXCL10/IP-10 in this in vivo model. Our results suggest that both CXCR3 and CXCL10/IP-10 contribute to resistance against primary dengue virus infection and that chemokines that are indistinguishable in in vitro assays differ in their activities in vivo.

Hemorrhage is a severe manifestation of dengue disease. Virus strain and host immune response have been implicated as the risk factors for hemorrhage development. To delineate the complex interplay between the virus and the host, we established a dengue hemorrhage model in immune-competent mice. Mice inoculated intradermally with dengue virus develop hemorrhage within 3 days. In the present study, we showed by the presence of NS1 antigen and viral nuclei acid that dengue virus actively infects the endothelium at 12 h and 24 h after inoculation. Temporal studies showed that beginning at day 2, there was macrophage infiltration into the vicinity of the endothelium, increased tumor necrosis factor alpha (TNF-␣) production, and endothelial cell apoptosis in the tissues. In the meantime, endothelial cells in the hemorrhage tissues expressed inducible nitric oxide synthase (iNOS) and nitrotyrosine. In vitro studies showed that primary mouse and human endothelial cells were productively infected by dengue virus. Infection by dengue virus induced endothelial cell production of reactive nitrogen and oxygen species and apoptotic cell death, which was greatly enhanced by TNF-␣. N G -Nitro-L-arginine methyl ester and N-acetyl cysteine reversed the effects of dengue virus and TNF-␣ on endothelial cells. Importantly, hemorrhage development and the severity of hemorrhage were greatly reduced in mice lacking iNOS or p47 phox or treatment with oxidase inhibitor, pointing to the critical roles of reactive nitrogen and oxygen species in dengue hemorrhage.