Development of an in-vitro dendritic cell model for studying dengue virus and host interaction

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Development of an in-vitro dendritic cell model for studying dengue virus and host interaction


Author: Zhang, Jingshu
Title: Development of an in-vitro dendritic cell model for studying dengue virus and host interaction
Degree: Ph.D.
Year: 2013
Subject: Dengue viruses.
Dendritic cells.
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Health Technology and Informatics
Pages: xxvii, 266 p. : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record:
Abstract: Dengue virus (DENV) infection is one of the most important arthropod-borne infections threatening populations in tropical and sub-tropical areas. The pathogen causes a mild flu-like illness, which may progress into life-threatening dengue haemorrhagic fever and dengue shock syndrome. So far, there is no specific treatment for dengue infections. Although dengue vaccines are undergoing different stages of clinical trials, the effectiveness remains to be assessed. Despite the immense research efforts on the pathogenesis of dengue virus, there are still a lot of virus-host interactions that remain unknown. The lack of suitable in-vivo animal models and in-vitro cell models is one of the obstacles to the understanding of dengue virus pathogenesis. Dendritic cells (DCs) are among the major targets of the DENV virus and are initiator of innate immune responses against DENV. However, current in-vitro research on the interaction between DENV-DC is hampered by the low availability of ex-vivo DCs and by donor variation. This project aimed to develop a novel in-vitro immature DC model derived from a myeloid leukaemia cell line MUTZ-3 for studying the DENV-DC interaction during type 2 dengue virus (DENV2) infection. The DC model derived from MUTZ-3 cells was compared to DCs derived from primary human monocytes in terms of the morphology, phenotypes, virus entry mechanism, permissiveness to DENV replication and antiviral pathway responses. In the current study, immature MUTZ-3-derived DCs (IMDCs) were shown to morphologically and phenotypically resemble immature human monocyte derived-DCs (IMMoDCs). However, RT-PCR arrays revealed that certain antiviral genes in IMDC, especially the interferon (IFN)-inducible genes such as IFIT1, IFITM1, and IFI27, had much higher expression levels than that of the IMMoDCs. When challenged with DENV2, it was found that the replication level of DENV2 in IMDCs was significantly lower than that in IMMoDCs. To better understand the causal relationship between the activated IFN-inducible genes and the reduced permissiveness in IMDCs, post-infection RT-PCR arrays on antiviral genes were conducted. It was found that the IFN-inducible genes, such as IFIT1, IFITM1, and IFI27, were significantly up-regulated in the DENV2-infected IMMoDCs but not in DENV2-infected IMDCs. Further investigation showed that protein expression levels of IFIT1 and IFIT3 were also higher in the naive IMDCs compared to that in the naive IMMoDCs. Similarly, DENV2 infection significantly triggered the expression of these two proteins in the IMMoDCs. It was suggested that spontaneous activation of these genes and the protein products might be important factors in the reduced permissiveness of IMDCs to DENV2.
DENV2 entry mechanism was also investigated using the established IMDC model. In the current study, DENV2 was shown to enter the immature DCs via clathrin-dependent endocytosis, while an alternative internalisation pathway was suggested because inhibiting the endocytosis pathway with inhibitors failed to completely block DENV2 entry. Two known DENV receptors, Dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) and mannose receptor (MR), were shown to co-express on approximately half of the populations of IMDCs and IMMoDCs. Blocking these two receptors at the same time efficiently inhibited DENV2 entry into IMDCs and IMMoDCs, even though the blockage was incomplete. These evidences together indicated that both DC-SIGN and MR were needed during DENV internalisation. Due to the fact that blocking of DENV2 receptors and inhibition of endocytosis could not completely block entry of DENV2, this study also investigated the possible involvement of other molecules during DENV2 entry. Using immunoprecipitation, the inter-{220}-trypsin inhibitor protein heavy chain 2 (ITIH2) was pulled down by the DENV2 virion from the transmembrane protein mixture, indicating an interaction of DENV2 with this molecule. Blocking ITIH2 together with DC-SIGN and MR significantly inhibited DENV2 entry to DCs. Thus, ITIH2 might be involved in DENV2 entry into its target cells. In summary, the IMDC model has been established and evaluated in this study. It was found that the IMDC model was similar with IMMoDCs in morphology and phenotype. The IMDC model was also found to express DC-SIGN and MR, which made it a useful platform for studying entry mechanisms of DENV2. Although IMDC had lower permissiveness to DENV2, when being used in parallel with the standard IMMoDC model, this novel IMDC model may help to expand the knowledge of DENV2 life-cycle, and of cellular factors that modulate DENV2 infection in the human body.

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