Identification of Pattern Recognition Receptors Interacting with Dengue Virus

• Main Authors: Ming-Ting Huang, 黃明停
• Other Authors: Shie-Liang Hsieh
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/61511297754711990778

碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 94 === Dengue is a vector-borne disease which causes mild dengue fever (DF) or severe dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), but the underlying molecular mechanism is still unknown. β-glucan can stimulate immune cells via dectin-1, a member of C-type lectin receptor, to secrete cytokines, and members of C-type lectins such as DC-SIGN and DC-SIGNR, can interact with dengue virus (DEN). To understand whether DEN could interact with other members of C-type lectin receptor, the extracellular domain of members of C-type lectins expressed on macrophages and dendritic cells were cloned and fused with IgG1mut to produce recombinant fusion proteins as probes for sandwich ELISA and immunoprecipitation. In addition to DC-SIGN, DC-SIGNR, hDVLR1 was found to interact with DEN serotype II (DEN-2) specifically, and knock-down of hDVLR1 blocked DEN-2-induced TNF-α-release from macrophage. To further confirm this observation, mice were immunized with recombinant hDVLR1.Fcmut to generate mAbs. Among the 66 anti-hDVLR1 mAbs, 9 clones could recognize hDVLR1 by flow cytometry, and further characterization found that clone 3E12, 8H8 and 9B12 can inhibit DEN-2-induced TNF-α release. Moreover, TNF-α was released after incubating macrophage with clone 3E12 (IgG1) and 8H8 (IgG1) in conjunction with goat anti-mouse IgG1 mAbs. To establish an in vivo model to test the role of DVLR1, we cloned the murine DVLR1 (mDVLR1), and alignment of hDVLR1 with mDVLR1 revealed that mDVLR1 is lack of 23 amino acids in the stalk region. Like hDVLR1, mDVLR1 is expressed on myeloid lineage and interacts with DEN-2, and DEN-2 can also stimulate murine macrophage-like cell line RAW 264.7 with MOI > 30, which is higher than incubating with human macrophages (MOI > 0.5), suggesting the 23 aa in stalk region might decrease the interaction between mDVLR1 and DEN-2. Knock down of mDVLR1 by shRNA suppressed DEN-2-induced TNF-α release from RAW 264.7, suggesting mDVLR1 also acts as a pattern recognition receptor to DEN-2. Thus, DVLR1 might be an ideal target to develop anti-inflammatory drugs for DHF and DSS in the future.