abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Mannose-binding lectin (MBL) functions as pattern recognition molecule to mediate first-line host defense against invading pathogens. Although MBL is well-known for its anti-bacterial action, its role towards virus infection is less comprehensively understood. In 2009, the pandemic H1N1 2009 (pdmH1N1) influenza A virus caused more than 18,000 deaths worldwide and is still circulating in human community as a seasonal strain. In this study, the role of MBL in pdmH1N1 infection was investigated. Using in vitro microtiter capture assay, MBL was found to bind to pdmH1N1 virus via its carbohydrate recognition domain. Under transmission electron microscope (TEM), MBL was clearly visible on the surface of pdmH1N1 virus. By infecting C57B6/J wild-type (WT) and MBL knockout (KO) mice with a sub-lethal dose of pdmH1N1 virus, WT mice displayed greater weight loss and more severe lung damage than MBL KO mice. Using flow cytometry-based profiling analysis of the lung homogenates isolated from infected mice, a variety of proinflammatory cytokines and chemokines were found to be significantly up-regulated. These results indicate that the presence of MBL can cause excess proinflammatory cytokine production and result in a more severe pdmH1N1 infection. To provide physiologically relevant insight into the immunomodulating role of MBL, the investigation was further extended to the use of human cell line model. Infection of A549 cells, which is a human lung epithelial cell line, with MBL-bound pdmH1N1 virus elevated the production of MCP1, RANTES and IL-8 significantly more than unbound pdmH1N1 infection. The increased production of chemokines also enhanced recruitment of monocytes as demonstrated by transwell migration assay. Interestingly, MBL did not affect viral entry or replication kinetics. TEM and confocal imaging revealed the presence of MBL-bound pdmH1N1 inside infected A549 cells, suggesting that the endocytosed MBL may interact with intracellular components to promote the release of cytokines and chemokines. To this end, expressions of Toll-like receptors were examined (TLR3, TLR7, TLR8 and TLR9) and found that TLR3 expression was dramatically enhanced upon pdmH1N1 infection. Interestingly, in MBL-bound pdmH1N1 infection, TLR3 mRNA and protein expression was significantly higher than unbound pdmH1N1 infection in A549 cells. In addition, the NF-κB signaling was further activated in the presence of MBL-bound pdmH1N1. A novel physical interaction between MBL and TLR3 was also delineated as evidenced by MBL’s capability to bind to TLR3 in vitro; and their colocalization in the endosomes of the infected A549 cells. In summary, MBL can bind to pdmH1N1 virus but fails to inhibit its infection in human lung epithelial cell line. Upon pdmH1N1 infection, MBL is internalized with the virus into the cell, where it may associate with TLR3 to further amplify the NF-κB signaling and augment the cytokine production in the human lung epithelial cells. The present findings advocate the adverse immunomodulating role of MBL during pdmH1N1 infection. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy