Effect Of HIV/HCV Co-infection On PDC-NK Cell Interaction

Objective To explore the interaction mechanism(s) of two kinds of natural immune cells: plasmacytoid dendritic cell (pDC) and Natural Killer (NK) cells;To investigate the effect of HIV/HCV mono-infection and co-infection on the interaction of pDC-NK cells, so as to to provide new information for the understanding of the interaction between HIV and HCV infections.Methods 1.Using pDC and NK cells from healthy subjects to study the interaction as well as mechanism(s) of pDC-NK cells : Firstly, using immunomagnetic separation to isolate pDC and NK cells, establish an in vitro pDC-NK cell co-culture system, confirm the expression of related cytokines(IFN-a, IFN-y, perforin and FasL, Granzyme-B) in activated pDC-NK cell system in vitro. ; Secondly, using transwell test to explore the role of direct cell-to-cell contactof pDC-NK cells in the activation of the cell system. 2.Using HIV/HCV-infected subjects (HIV mono-infection, HCV mono-infection,HIV/HCV co-infection) and healthy control subjects to study the effect of virus infection on the interaction of pDC-NK cells: after collecting the blood samples from virus infected people, the pDC-NK cell activation tests were carried out as described above in different groups, through comparing with the healthy group,the effects of virus infection on the pDC-NK cell system were analyzed.Results1. Activation mechanism of pDC-NK cell system:1.1 pDC cells can be activated in vitro and release a large number of IFN-?.After adding TLR-9 receptor agonist ODN2216 to the freshly isolated pDC, the expression of IFN-? in the treatment group was significantly higher than that in the standard control group (ODN2243 treatment). The difference was statistically significant (P<0.01).1.2 in vitro NK cells can be activated by the ODN2216-activated pDC(through activation of TLR-9 pathway), and express related cytokines. Freshly isolated NK cells were directly treated with ODN2216, and the results show that the expression of NK cell related cytokines (IFN-?, Perforin, Fasl, Granzyme-B)at mRNA level did not change significantly (P> 0.05). After adding a small amount of pDC cells (pDC: NK cells=1:10), the expression of the NK cell related cytokines (IFN-??Perforin?Fasl?Granzyme-B) at the mRNA level was significantly higher than that of t the standard control( P <0.05).1.3 Direct contact between pDC and NK cells is not a major factor of the activation of NK cells. Based on the results of the above experiments, the transwell test was carried out. pDC and NK cells can be cultured separately under the conditions without direct contact but share culturesolution. The results show that without pDC-NK direct contact, NK cells can be still activated (the expression of perforin and FasL, Granzyme-B was up-regulated, compared with standard control group, P<0.05). The results of transwell co-culture group were compared with direct contact co-culture group (not using transwell), showing that there is no significant difference in the expression of NK cell related cytokines.2. The influence of virus infections (HIV/HCV) on pDC-NK cell system2.1 HIV infection can up-regulate the expression of the pDC background IFN-a expression at mRNA level. The expression of IFN- ? at mRNA level in freshly isolated pDC from HIV-infected patients was compared with that from the healthy control group, showing there is a significant increase in IFN-aexpression (P<0.01). But the expression of IFN-? at mRNA level in ODN2216-treated pDC from HIV-infected subjects is significantly lower than that fromhealthy control group (P<0.01), suggesting that HIV infection damage the in vitro activation of pDC by TLR-9 pathway..Meanwhile, the expression of Granzyme-B mRNAin ODN2216-treated pDC-NK cell system from HIV-infected group is significantly higher than that of healthy control group (p<0.01).2.2 HCV can also up-regulate the background expression of IFN-? at mRNA level in freshly isolated pDC. HCV infection can also promote the ability of ODN2216-activated pDC to express higher level of IFN-? at mRNA level,compared with that of the ODN2216-activated pDC from healthy control subjects. (474.53±10.62vs249.08±28.26)(p<0.01).The background expression of some cytokines from pDC-NK cell system from HCV-inefcted subjects was down-regulated, compared with those from healthy control subjects (IFN-y: 0.44±0.51, Fasl: 0.52±0.28). But the expression of Granzyme-B at mRNA level was up- regulated. HCV infection affects the interaction of pDC-NK cell system.2.3 HIV/HCV coinfection can also up-regulate the IFN-? mRNA background expression of freshly isolated pDC. Meanwhile, HIV/HCV co-infection can improve the background expression of NK cell related cytokines in pDC-NK cell system.Conclusion This study has successfully established an in vitro pDC-NK cell culture system. In this system, NK cells can be activated by ODN2216-activated pDC, as evidenced by induced expression of a variety of cytokines secreted from NK cells. The present study indicates that the interaction between pDC-NK cells is not triggered by direct cell-to-cell contact,suggesting that cytokines produced from pDC plays an important role in interaction of pDC-NK cells. This study further explored the effects of HIV infection, HCV infection and HIV/HCV infection on the pDC-NK cell system.The results show that HIV and HCV monoinfection as well as HIV/HCV co-infection can cause in vivo pDC cells in the activated state. However, HIV monoinfection can damage in vitro TLR-9 pathway activation of pDC, and thus impair pDC-NK cell interaction. HCV monoinfection, in the contrary, can enhance the pDC TLR-9 pathway activation in vitro, thereby affect the mutual effect pDC-NK interaction. For HIV/HCV co-infection, the activation of pDC-NK cell system in vitro is similar to that of HCV monoinfection,HIV/HCV co-infection can also promote the activation of pDCin vitro, and then affect the interaction of pDC-NK cells.