Doris Wilflingseder

HIV-1-Induced Migration of Monocyte-Derived Dendritic Cells Is Associated with Differential Activation of MAPK Pathways

From the site of transmission at mucosal surfaces, HIV is thought to be transported by DCs to lymphoid tissues. To initiate migration, HIV needs to activate DCs. This activation, reflected by intra- and extracellular changes in cell phenotype, is investigated in the present study. In two-thirds of the donors, R5- and X4-tropic HIV-1 strains induced partial up-regulation of DC activation markers such as CD83 and CD86. In addition, CCR7 expression was increased. HIV-1 initiated a transient phosphorylation of p44/p42 ERK1/2 in iDCs, whereas p38 MAPK was activated in both iDCs and mDCs. Up-regulation of CD83 and CD86 on DCs was blocked when cells were incubated with specific p38 MAPK inhibitors before HIV-1-addition. CCR7 expression induced by HIV-1 was sufficient to initiate migration of DCs in the presence of secondary lymphoid tissue chemokine (CCL21) and MIP-3β (CCL19). Preincubation of DCs with a p38 MAPK inhibitor blocked CCR7-dependent DC migration. Migrating DCs were able to induce infection of autologous unstimulated PBLs in the Transwell system. These data indicate that HIV-1 triggers a cell-specific signaling machinery, thereby manipulating DCs to migrate along a chemokine gradient, which results in productive infection of nonstimulated CD4+ cells.

Complement dependent trapping of infectious HIV in human lymphoid tissues.

Objectives:HIV-1 bound extracellularly to follicular dendritic cells (FDC) in germinal centers (GC) of lymphoid tissues (LT) represents the largest viral reservoir in HIV-infected individuals; however there is no direct evidence as to whether HIV trapped in human GC remains infectious. In the GC, complement receptors and Fcγ receptors have been suggested to participate in trapping of HIV; therefore, the relative contribution of complement- and Fcγ receptors in binding HIV on LT was investigated and the infectivity of this virus was tested. Design:As it is difficult to isolate FDC without contaminations of productively infected cells, HIV was detached from LT of HIV positive individuals using antibodies blocking complement- and Fcγ receptors. Isolated virus was tested in an infectivity assay. Methods:HIV detached from LT was quantified by HIV p24 ELISA, PCR and in an in vitro infectivity assay. Presence and accessibility of viral envelope proteins, complement factors and immunoglobulins on the surface of detached viral particles were evaluated through viral capture by respective antibodies. Results:Although both C3d-fragments and IgG molecules were identified on the surface of HIV detached from LT, trapping of HIV was mediated solely by CR2–C3d interactions, whereas contribution of Fcγ receptors was not detectable. Infectivity assays with HIV stripped from LT of HIV positive individuals revealed that in four out of ten patients HIV particles were infectious. Conclusions:These findings indicate that in the GC infectious virus is trapped on CR2-expressing FDC (or B cells). Reduction of this pool of HIV could be a therapeutic goal.

Cross-Linking of CD32 Induces Maturation of Human Monocyte-Derived Dendritic Cells Via NF-κB Signaling Pathway

Dendritic cells (DC) represent a unique set of APCs that initiate immune responses through priming of naive T cells. Maturation of DC is a crucial step during Ag presentation and can be induced by triggering a broad spectrum of DC surface receptors. Although human DC express several receptors for the Fc portion of IgG which were described to play an important role in Ag internalization, little is known about the effects of IgG or immune complexes on DC maturation. In this study, we show that cross-linking of FcγR-type II (CD32) with immobilized IgG (imIgG) can induce maturation of human monocyte-derived DC via the NF-κB signaling pathway. IgG-mediated maturation was accompanied by a moderate increase of IL-10 secretion, whereas no IL-12 production was observed. Involvement of CD32 was further supported by experiments with the anti-CD32 mAb, which blocked IgG-triggered DC maturation and cytokine secretion significantly. Furthermore, DC cultivated in the presence of imIgG induced allogeneic T cell proliferation. Because this imIgG-induced maturation was considerably impaired in monocyte-derived DC from systemic lupus erythematosus patients, we suggest that DC, which matured in the presence of immune complexes, may contribute to prevention of pathological immune responses.

Complement-HIV interactions during all steps of viral pathogenesis

Upon crossing the endothelial barrier of the host, HIV initiates immediate responses of the immunity system. Among its components, the complement system is one of the first the first elements, which are activated to affect HIV propagation. Complement participates not only in the early phase of the immune response, but its effects can be observed continuously and also concern the induction and modification of the adaptive immune response. Here we discuss the role of complement in early and late stages of HIV pathogenesis and review the escape mechanisms, which protect HIV from destruction by the complement system.

Pseudotyping vesicular stomatitis virus with lymphocytic choriomeningitis virus glycoproteins enhances infectivity for glioma cells and minimizes neurotropism

ABSTRACT Vesicular stomatitis virus (VSV)-based oncolytic virotherapy has the potential to significantly improve the prognosis of aggressive malignancies such as brain cancer. However, VSVs inherent neurotoxicity has hindered clinical development so far. Given that this neurotropism is attributed to the glycoprotein VSV-G, VSV was pseudotyped with the nonneurotropic envelope glycoprotein of the lymphocytic choriomeningitis virus (LCMV-GP→VSV-GP). Compared to VSV, VSV-GP showed enhanced infectivity for brain cancer cells in vitro while sparing primary human and rat neurons in vitro and in vivo, respectively. In conclusion, VSV-GP has a much wider therapeutic window than VSV and is thus more suitable for clinical applications, especially in the brain.

Minireview: host defence in invasive aspergillosis.

Aspergillus is a saprophytic fungus, which mainly becomes pathogenic in immunosuppressed hosts. A failure of host defences results in a diverse set of illnesses, ranging from chronic colonisation, aspergilloma, invasive disease and hypersensitivity. A key concept in immune responses to Aspergillus species is that host susceptibility determines the morphological form, antigenic structure and physical location of the fungus. Traditionally, innate immunity has been considered as a first line of defence and activates adaptive immune mechanisms by the provision of specific signals; innate and adaptive immune responses are intimately linked. The T‐helper cell (TH1) response is associated with increased production of inflammatory cytokines IFN‐γ, IL‐2 and IL‐12 and stimulation of antifungal effector cells. Alternatively, TH2‐type responses are associated with suppression of antifungal effector cell activity, decreased production of IFN‐γ and increased concentrations of IL‐4 and IL‐10, which promote humoral responses to Aspergillus. The host’s defensive capacity is defined by the sum of resistance and tolerance. Resistance displays the ability to limit fungal burden and elimination of the pathogen, and tolerance means the ability to limit host damage caused by immune response.

C-type lectin-independent interaction of complement opsonized HIV with monocyte-derived dendritic cells

HIV directly activates the complement cascade and is, therefore, opsonized with C3‐cleavage products in vivo. This cloud of C3 fragments on the viral surface may impair the interaction of the HIV envelope glycoproteins gp120/gp41 with C‐type lectins expressed on immature dendritic cells (iDC). Therefore, we determined the accessibility of gp120 after opsonization and compared the interaction of DC with non‐opsonized or complement‐opsonized HIV. The recognition of native gp120 was drastically impaired when the virus was covered by complement. Independent of opsonization, similar amounts of HIV bound to DC. The interaction of iDC and the infection of DC‐PBL co‐cultures with non‐opsonized virus was significantly reduced by mannan and antibodies which inhibit the ICAM‐1‐CR3 interaction. The binding of opsonized virus to iDC was reduced by an anti‐CR3‐antibody, which interferes with the binding of C3 fragments, but was not affected by mannan. Complement enhanced the HIV infection of DC and DC‐PBL co‐cultures significantly. Mannan did not inhibit the complement‐dependent enhancement of infection. Thus, non‐opsonized and opsonized HIV interacted with iDC, although the binding mechanisms seemed to differ. As HIV is opsonized in vivo, the C‐type lectin‐independent interaction of opsonized viruses with iDC has to be taken into account.

IgG Opsonization of HIV Impedes Provirus Formation in and Infection of Dendritic Cells and Subsequent Long-Term Transfer to T Cells

Already at initial phases of infection, HIV is coated with complement fragments. During the chronic phase, when HIV-specific IgGs appear, the virus circulates immune complexed with IgG and complement. Thus, we studied the interaction of dendritic cells (DCs) and DC-T cell cocultures with complement (C)-opsonized and C-IgG-opsonized HIV. HIV infection of monocyte-derived DCs and circulating BDCA-1-positive DCs was significantly reduced upon the presence of virus-specific but non-neutralizing IgGs. DCs exposed to C-Ig-HIV or IgG-opsonized HIV showed an impaired provirus formation and p24 production and a decreased transmission rate to autologous nonstimulated T cells upon migration along a chemokine gradient. This reduced infectivity was also observed in long-term experiments, when T cells were added delayed to DCs exposed to IgG-coated HIV without migration. Similar kinetics were seen when sera from HIV-1-infected individuals before and after seroconversion were used in infection assays. Both C- and C-IgG-opsonized HIV were captured and targeted to a tetraspanin-rich endosome in immature DCs, but differed with respect to MHC class II colocalization. The reduced infection by IgG-opsonized HIV is possibly due to interactions of virus-bound IgG with FcγRIIb expressed on DCs. Therefore, the intracellular fate and transmission of immune-complexed HIV seems to differ depending on time and opsonization pattern.

Mechanism(s) promoting HIV-1 infection of primary unstimulated T lymphocytes in autologous B cell/T cell co-cultures

Resting CD4+ T cells in the lymphoid tissue (LT) are essential producers of virions at the beginning of HIV infection in vivo. We previously developed a model that allowed in vitro infection of non‐prestimulated T lymphocytes in the presence of autologous B lymphocytes and complement. In this study, we try to clarify the mechanism(s) responsible for virus transmission in unstimulated autologous B cell/T cell co‐cultures. Ex vivo analyses of patient plasma samples revealed that HIV was opsonized. Flow cytometry showed that opsonized virus preferentially bound to complement receptor (CR)‐2 on B lymphocytes in primary B cell/T cell co‐cultures. As indicated by cytokine measurements and transwell experiments, soluble factors seemed to play a minor role in enabling infection. Rather, direct interaction between B and T lymphocytes and direct binding of opsonized virus to CR2 on B cells turned out to be essential for productive infection. Antibodies blocking cell‐cell adhesion inhibited p24 antigen production. An anti‐CR2 antibody blocking C3d‐CR2 binding also significantly reduced viral replication. Since the infection of unstimulated T cells by opsonized primary HIV isolates in the presence of B cells was highly efficient independent of the tropism of the virus, this mechanism may be critical in the pathogenesis of HIV.

Factor I-Mediated Processing of Complement Fragments on HIV Immune Complexes Targets HIV to CR2-Expressing B Cells and Facilitates B Cell-Mediated Transmission of Opsonized HIV to T Cells

Our study demonstrates that binding of complement-opsonized HIV to complement receptor type 1 on human erythrocytes (E) via C3b fragments is followed by a rapid normal human serum-mediated detachment of HIV from E. The release was dependent on the presence of factor I indicating a conversion of C3b fragments to iC3b and C3d on the viral surface. This in turn resulted in an efficient binding of opsonized HIV to CR2-expressing B cells, thus facilitating B cell-mediated transmission of HIV to T cells. These data provide a new dynamic view of complement opsonization of HIV, suggesting that association of virus with E might be a transient phenomenon and the factor I-mediated processing of C3b to iC3b and C3d on HIV targets the virus to complement receptor type 2-expressing cells. Thus, factor I in concert with CR1 on E and factor H in serum due to their cofactor activity are likely to be important contributors for the generation of C3d-opsonized infectious HIV reservoirs on follicular dendritic cells and/or B cells in HIV-infected individuals.