Study of Immunopathogenesis of Liver Damage and Hemorrhage in Dengue Mouse Models
Date Issued
2007
Date
2007
Author(s)
Chen, Hsuen-Chin
DOI
en-US
Abstract
Dengue is one of the most important mosquito-borne diseases in the world. In the past 50 years, its incidence has increased 30-fold with significant outbreaks occurring in tropical and sub-tropical regions of the world. Dengue virus (DV) causes a wide range of disease in humans, from dengue fever (DF) to life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The pathogenesis of severe dengue disease is not well understood. So far, there is no suitable animal model to study the immunopathogenesis of dengue. Therefore, to develop mouse model is necessary and urgent to the understanding of the pathogenesis of dengue.
Evidences show that dengue virus-specific T cells are activated and expanded in patients with dengue and the extent of T cell activation correlates with development of severe disease. Elevated liver enzyme levels in the sera of patients with severe dengue disease have been documented, indicating that dengue patients have abnormal liver functions. However, it remains to be investigated whether T cells are involved in the pathogenic mechanism of liver injury in dengue. In the first part of my study, immunocompetent C57BL/6 mice were inoculated intravenously with 108 PFU DV serotype 2 strain 16681. Dengue viral capsid RNA was detectable in the serum, liver, spleen, and brain by RT-PCR at different time points after infection. Splenic B and T cells are activated as evidenced by the expression of CD69 at as early as day 1 of infection. Activated T cells also expressed O-glycosylated CD43 at day 3 of infection. T cell expression of O-glycosylated CD43 and IFN-γ 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 that infiltrating cells included CD8 and CD4 T cells and their ratio was 5 to 3. Hepatic T cell infiltration and liver enzyme levels increased sharply at day 3 after second noculation of DV. The results demonstrated a strong correlation between T cell activation, hepatic cellular infiltration, and elevation of liver enzymes in mice infected with DV. It implied that activated T cells may be pathogenic to the host and cause liver injury in DV infection.
Hemorrhage is a common clinical manifestation in dengue patients. Thepathogenic mechanism of DV-induced hemorrhage still awaits clarification. In the second part of my study, a dengue hemorrhage mouse model was established in
immunocompetent C57BL/6 mice by intradermal inoculation of DV-2 strain 16681. While inoculation of 3 × 109 PFU of DV induced systemic hemorrhage in all of the mice by day 3 of infection, 1/3 of those injected with 4-8 × 107 PFU developed hemorrhage in the subcutaneous tissues. H & E stain of the hemorrhagesubcutaneous tissue sections revealed signs of vascular leakage. The mice that wereinoculated with 4-8 × 107 PFU but did not develop hemorrhage were used as a basisfor comparison to explore the pathogenic mechanism of dengue hemorrhage. Itappeared that high viral titer with in 1-3 days after infection had positive correlationwith hemorrhage development. Results showed that the platelet counts weresignificantly reduced in both hemorrhage and non hemorrhage mice at 3 days afterinfection. The counts were further reduced in hemorrhagic mice but remained at thesame level as that on day 3 in nonhemorrhage mice at day 7. At day 3, the time ofhemorrhage development, there was massive macrophage infiltration, high level ofTNF-α production, and overwhelming presence of apoptotic cells in the hemorrhagetissue. Immunofluorescence staining revealed that DV targeted both endothelialcells and macrophages became apoptotic, and >90% of the endothelial cells wereapoptotic. Taken together, high viral titer, macrophage infiltration, TNF-αproduction in the local tissues are three important events that lead to hemorrhage. In addition,production of high TNF-α in tissues correlated with endothelial cell apoptosis and hemorrhage.
At the time of hemorrhage development, there was also deposition of complement 3 in the hemorrhage tissue, DV-specific antibody production and lymph node T cell activation. To clarify the role of TNF-α and the involvement of other immune factor(s) in the immunopathogenesis of DV-induced hemorrhage, I infected TNF-α -/- to TCRβ-/-, IgH-/-, STAT1-/-, IFN-γ-/-, A/HeJ (C5-/-) mice with DV and ompared to wild type mice for hemorrhage development. The percentage of immune deficient mice that developed hemorrhage was not different from the wild type mice except those with TNF-α deficiency. This finding indicates that TNF-α but not other immune factors is important to hemorrhage development.
In vitro studies showed that primary mouse microvascular endothelial cells were susceptible to DV and TNF-α enhanced DV-induced apoptosis. Supernatants from DV-infected macrophage cultures also enhanced DV-induced endothelial cell death and anti-TNF-α antibody treatment abolished the effect. These results demonstrated
that DV infection of macrophages induces TNF-α production and TNF-α enhanced DV-induced endothelial cell death. The mouse model established in the present study illustrates that intradermal inoculation of high titers of DV predisposes endothelial cells to TNF-α-induced cell death which leads to endothelium damage and hemorrhage development. This finding highlights the contribution of innate immune response to dengue hemorrhage.
In summary, I have successfully established two mouse models to study the immunopathogenic mechanisms of DV-induced liver damage and hemorrhage. Each of them will be useful for further dissection of the pathogenic mechanisms of immune response to disease development in hosts infected by dengue virus.
Subjects
Dengue
animal model
liver damage
hemorrhage
SDGs
Type
other
File(s)![Thumbnail Image]()
Loading...
Name
ntu-96-F87449001-1.pdf
Size
23.31 KB
Format
Adobe PDF
Checksum
(MD5):6fa0b963a8175688092ac7aa1dc6ea57