Human dendritic cells and hepatitis C Virus
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Dendritic cells (DCs) constitute a large family of immune cells with a dendritic morphology and a critical role in all aspects of an immune response and immune regulation, from immunogenicity to tolerance. One of the important characteristic of DCs is maturation, during which DCs undergo significant changes in their phenotypic and functional properties and change from phagocytic cells to highly efficient antigen presenting cells (APCs). Dendritic cells have recently been at the centre of attention as a promising tool in treatment or control of cancer and infectious diseases. Accordingly, DCs have been generated, matured, and loaded with tumor-associated or microbial antigens ex vivo, to be subsequently used as therapeutic tools or vaccine carriers. Hepatitis C virus (HCV) is a hepatotropic virus, which infects the liver in humans and results in a chronic infection in most cases. The persistent infection of the liver eventually results in cirrhosis and/or hepatocellular carcinoma in 15-20 years. Chronic hepatitis C (CHC) has recently become a serious health concern and the leading cause of liver transplantation. The mechanism of persistence of the virus is not clear yet, but as a Th1-type immune response is strongly correlated with elimination of HCV in vivo, it is evident that insufficient cellular immunity is a contributing factor. Non-cytopathic viruses such as HCV may infect immune cells to modify and evade a protective immune response. Dendritic cells, which are the most potent APCs, and uniquely capable of initiating a primary immune response, have been considered as a target for HCV. Inhibition of DC maturation by HCV has been suggested as a potential contributing factor in immune evasion; however, this issue remains controversial as many contradictory results have been reported. To investigate this contention, we initially planned to evaluate the effects of HCV on DCs of CHC patients; however, due to limited access to patients’ blood, we instead elected to examine the effects of HCV genes products on in vitro generated DCs from healthy volunteers. Specific attention was paid to the generation, maturation, and transfection of DCs in vitro, as variability in procedures might have been responsible for the controversial reports. Viral vectors have generally been used to transfect DCs; however, a vector and HCV genes might have synergistic effects on DC maturation. Thus, our first objective was to develop an efficient non-viral transfection method while retaining high viability of the DCs, as previous efforts in this regard resulted either in low efficiency or in low viability of DCs after transfection. In order to improve the viability of DCs after transfection, we established a new method for fast generation of monocyte-derived DCs (Mo-DCs) in two to three days. By performing a comprehensive study on transfection reagents, electroporation, and nucleofection with DNA or in vitro transcribed (IVT) RNA, we successfully established a new, highly efficient non-viral method for transfection of DCs with long-term viability. This method is based on the use of the X1 program of a nucleofection device with IVT RNA and results in high transfection efficiency of 93%, with 75% viability of DCs 72 h after transfection. Subsequently, we performed a comprehensive study on the effects of different maturation methods on the phenotype, function and gene expression profile of DCs. Three commonly used treatments, TNF-α, LPS and a maturation cocktail (MC) consisting of IL-1β, IL-6, TNF-α, and prostaglandin E2 (PGE2) were compared. Our results showed that there is a significant difference in the level of maturity between these treatments, and MC generated more functionally competent mDCs than TNF-α or LPS. In addition, MC induced Th1-promoting changes in the transcriptional profile of mDCs. This observation was important, as the presence of PGE2 in MC was previously challenged based on the potential induction of Th2-biased immune responses. However, our results suggest retaining PGE2 in the cocktail because of the fact that MC generated highly competent and functional mDCs with a Th1-promoting transcriptional profile. Finally, Mo-DCs were transfected with IVT HCV RNAs, individually or in combination. While HCV genes had no inhibitory effect on DC maturation, transfection of DCs with IVT core RNA appeared to result in changes compatible with maturation. To investigate this in more detail, the transcriptional profiles of DCs transfected with IVT core, NS3 or green fluorescent protein (GFP) RNA were examined using a DC-specific membrane array. Of the 288 genes on the array, 46 genes were distinctively up- or down-regulated by transfection with IVT core RNA in comparison to NS3 or GFP RNA treatments, 42 of which are involved in DC maturation. The effects of core on maturation of DCs were further confirmed by a significant increase in surface expression of CD83 and HLA-DR, a reduction of phagocytosis, as well as an increase in proliferation and IFN-γ secretion by T cells in a mixed lymphocyte reaction assay. These results show that HCV core does not have an inhibitory effect on human DC maturation, but could be a target for the immune system. The use of a non-viral method of transfection combined with confirmed transcriptional profiles of DCs in this study may make these results conclusive for in vitro generated DCs from healthy volunteers. However, further investigations are required to confirm the effects on DCs from CHC patients.
DegreeDoctor of Philosophy (Ph.D.)
Supervisorvan den Hurk, Sylvia
CommitteeBabiuk, Lorne A; Qualtiere, Louis; Williams, Kurt; Misra, Vikram; Elliott, John F
Copyright DateDecember 2009
hepatitis C virus