Role of complement in HIV infection of dendritic cells

The goal of this project is to further elucidate the role of complement in the earliest phases of HIV infection, particularly in the process of virus transmission and spread via DC. Since more than a decade, our laboratory is engaged in the field of the complement system and its interaction with HIV. With financial support by the Austrian Ministry of Science (BMBWK), good-working collaborations with institutes from "EU-associating countries" Hungary and Slovakia have been established. We would like to exploit this fruitful research cooperation to further investigate the role of complement in the course of HIV pathogenesis. To achieve this goal, we plan to pursue the following five specific aims:First, we plan to investigate the role of complement receptors (CR) and their ligands, as well as other potential molecules, in the process of HIV attachment to DC. For this purpose, virus stocks generated in vitro will be opsonized with immunoglobulins and complement fragments to obtain diverse opsonin patterns on the surface of virions and the contribution of particular CR to HIV binding will be assessed. Second, the effects of virus opsonization on HIV infection of DC will be studied. This part of the project should focus on dissimilarities between opsonized and non-opsonized virus stocks regarding the ability to productively infect DC in vitro. Third, to elucidate the mechanism by which DC transmit HIV to CD4+ target cells, the potential role of CR in this process will be investigated. For this purpose, DC will be loaded with opsonized and non-opsonized virus stocks and cultured in the presence of CD4+ cells. In addition, HIV transmission from DC into tonsillar tissue cultivated ex vivo will be studied in this system. Fourth, we will assess the secretion of cytokines from DC, cultivated in the presence or absence of monoclonal antibodies (mAb) directed against CR. Kinetics and cytokine pattern should be determined on both mRNA and protein level. Finally, these data will be conclusively correlated with HIV infection experiments. Fifth, we have shown recently that C5a and its desArg metabolite enhance HIV replication in monocyte-derived macrophages. Therefore, we will test whether complement-derived anaphylatoxins influence the interaction of HIV with DC as well.

Mucosal transmission represents the major route of primary HIV infections. Dendritic cells (DCs) presented at the mucosal surface are thought to be one of the first target cells for HIV infection. Since DCs express different complement receptors, like CR3 and CR4, the role of complement in the earliest phases of HIV infection, particularly in the process of virus transmission and spread via DCs was investigated in this project. In our study, we have shown that complement activation by HIV resulted in the generation of sufficient amount of complement anaphylatoxin C5a. C5a generated by HIV in the presence of normal human serum (NHS) induced DC-migration in trans-well experiments via C5a-receptors on DCs suggesting that C5a may account for the mobilization of iDCs to the site of viral entry at the beginning of infection and during initial phases of the pathogenesis. On the other hand, complement activation by HIV leads to the deposition of C3-fragments on the viral surface. This cloud of complement proteins on HIV impaired the accessibility of certain viral epitopes suggesting that opsonisation of HIV might influence DC-SIGN-dependent interactions of HIV with DCs. Indeed, the binding of non-opsonised HIV was inhibited by mannan, which blocks interactions of HIV with DC-SIGN and other C-type lectins, whereas complement-opsonised HIV interacted with DCs mainly through complement receptors. More importantly, we found a 2-3 fold increase in the infection of DCs with opsonised HIV when compared to non-opsonised virus. Simmilarly to the binding experiments, mannan was able to abrogate infection of DC by non-opsonised virus, whereas it did not affect the infection of DC by complement-opsonised HIV. These results suggest that both non- opsonised and complement-opsonised HIV can interact with dendritic cells, however both the binding and the infection was mediated by different receptors. In further experiments, DC-mediated transmission of HIV and C- HIV to autologous primary T cells was studied in co-culture experiments. According to previous reports, we found that DC-mediated transmission of non-opsonised HIV to T cells was mediated by C-type lectins. The infection of DC-T cell co-cultures with non-opsonized virus was significantly reduced by mannan. Complement-opsonisation enhanced the HIV infection of DC-T cell co-cultures significantly. In contrast to non-opsonised HIV, transmission of complement-opsonised HIV by DC was not influenced by mannan suggesting a C-type lectin independent mechanism in DC-mediated transmission of opsonised HIV. Taken together, our results suggest that complement activation by HIV might trigger DC-infection and DC-mediated virus dissemination in vivo.