Manja Litjens

Carbohydrate-specific signaling through the DC-SIGN signalosome tailors immunity to Mycobacterium tuberculosis, HIV-1 and Helicobacter pylori

Cooperation between different innate signaling pathways induced by pattern-recognition receptors (PRRs) on dendritic cells (DCs) is crucial for tailoring adaptive immunity to pathogens. Here we show that carbohydrate-specific signaling through the C-type lectin DC-SIGN tailored cytokine production in response to distinct pathogens. DC-SIGN was constitutively associated with a signalosome complex consisting of the scaffold proteins LSP1, KSR1 and CNK and the kinase Raf-1. Mannose-expressing Mycobacterium tuberculosis and human immunodeficiency virus type 1 (HIV-1) induced the recruitment of effector proteins to the DC-SIGN signalosome to activate Raf-1, whereas fucose-expressing pathogens such as Helicobacter pylori actively dissociated the KSR1–CNK–Raf-1 complex from the DC-SIGN signalosome. This dynamic regulation of the signalosome by mannose- and fucose-expressing pathogens led to the enhancement or suppression of proinflammatory responses, respectively. Our study reveals another level of plasticity in tailoring adaptive immunity to pathogens.

HIV-1 exploits innate signaling by TLR8 and DC-SIGN for productive infection of dendritic cells

Pattern-recognition receptors (PRRs) elicit antiviral immune responses to human immunodeficiency virus type 1 (HIV-1). Here we show that HIV-1 required signaling by the PRRs Toll-like receptor 8 (TLR8) and DC-SIGN for replication in dendritic cells (DCs). HIV-1 activated the transcription factor NF-κB through TLR8 to initiate the transcription of integrated provirus by RNA polymerase II (RNAPII). However, DC-SIGN signaling was required for the generation of full-length viral transcripts. Binding of the HIV-1 envelope glycoprotein gp120 to DC-SIGN induced kinase Raf-1–dependent phosphorylation of the NF-κB subunit p65 at Ser276, which recruited the transcription-elongation factor pTEF-b to nascent transcripts. Transcription elongation and generation of full-length viral transcripts was dependent on pTEF-b-mediated phosphorylation of RNAPII at Ser2. Inhibition of either pathway abrogated replication and prevented HIV-1 transmission. Thus, HIV-1 subverts crucial components of the immune system for replication that might be targeted to prevent infection and dissemination.

Salp15 binding to DC-SIGN inhibits cytokine expression by impairing both nucleosome remodeling and mRNA stabilization

Ixodes ticks are major vectors for human pathogens, such as Borrelia burgdorferi, the causative agent of Lyme disease. Tick saliva contains immunosuppressive molecules that facilitate tick feeding and B. burgdorferi infection. We here demonstrate, to our knowledge for the first time, that the Ixodes scapularis salivary protein Salp15 inhibits adaptive immune responses by suppressing human dendritic cell (DC) functions. Salp15 inhibits both Toll-like receptor- and B. burgdorferi–induced production of pro-inflammatory cytokines by DCs and DC-induced T cell activation. Salp15 interacts with DC-SIGN on DCs, which results in activation of the serine/threonine kinase Raf-1. Strikingly, Raf-1 activation by Salp15 leads to mitogen-activated protein kinase kinase (MEK)-dependent decrease of IL-6 and TNF-α mRNA stability and impaired nucleosome remodeling at the IL-12p35 promoter. These data demonstrate that Salp15 binding to DC-SIGN triggers a novel Raf-1/MEK-dependent signaling pathway acting at both cytokine transcriptional and post-transcriptional level to modulate Toll-like receptor–induced DC activation, which might be instrumental to tick feeding and B. burgdorferi infection, and an important factor in the pathogenesis of Lyme disease. Insight into the molecular mechanism of immunosuppression by tick salivary proteins might provide innovative strategies to combat Lyme disease and could lead to the development of novel anti-inflammatory or immunosuppressive agents.

Specific ICAM-3 grabbing nonintegrin-related 1 (SIGNR1) expressed by marginal zone macrophages is essential for defense against pulmonary Streptococcus pneumoniae infection

The dendritic cell‐specific ICAM‐3‐grabbing nonintegrin (DC‐SIGN) homolog, SIGN‐related 1 (SIGNR1) is a pathogen receptor expressed by splenic marginal zone and peritoneal macrophages, and is essential for clearance of Streptococcus pneumoniae by phagocytosis after intraperitoneal infection. Here, we identified an important in vivo function for SIGNR1 in S. pneumonia infection induced via its natural entrance route. Upon intranasal infection with S. pneumoniae, SIGNR1‐deficient mice did not clear bacteria from lung and blood, and displayed severely enhanced inflammatory parameters compared to the wild‐type mice. However, SIGNR1 is not expressed by alveolar macrophages, suggesting that another mechanism than a decrease in phagocytosis is responsible for this difference. Natural anti‐phosphorylcholine IgM produced by marginal zone B cells is essential for protection against infection with S. pneumoniae. Strikingly, during infection, SIGNR1‐deficient mice failed to produce a rapid anti‐phosphorylcholine IgM response. Marginal zone macrophages have been suggested to capture antigens for presentation to marginal zone B cells. We demonstrate that marginal zone macrophages from SIGNR1‐deficient mice in contrast to wild‐type mice are not able to capture pneumococci from blood, suggesting that SIGNR1 on marginal zone macrophages captures S. pneumoniae for antigen presentation to and activation of marginal zone B cells, resulting in an anti‐phosphorylcholine IgM response.

DC-SIGN and CD150 have distinct roles in transmission of measles virus from dendritic cells to T-Lymphocytes

Measles virus (MV) is among the most infectious viruses that affect humans and is transmitted via the respiratory route. In macaques, MV primarily infects lymphocytes and dendritic cells (DCs). Little is known about the initial target cell for MV infection. Since DCs bridge the peripheral mucosal tissues with lymphoid tissues, we hypothesize that DCs are the initial target cells that capture MV in the respiratory tract and transport the virus to the lymphoid tissues where MV is transmitted to lymphocytes. Recently, we have demonstrated that the C-type lectin DC-SIGN interacts with MV and enhances infection of DCs in cis. Using immunofluorescence microscopy, we demonstrate that DC-SIGN+ DCs are abundantly present just below the epithelia of the respiratory tract. DC-SIGN+ DCs efficiently present MV-derived antigens to CD4+ T-lymphocytes after antigen uptake via either CD150 or DC-SIGN in vitro. However, DC-SIGN+ DCs also mediate transmission of MV to CD4+ and CD8+ T-lymphocytes. We distinguished two different transmission routes that were either dependent or independent on direct DC infection. DC-SIGN and CD150 are both involved in direct DC infection and subsequent transmission of de novo synthesized virus. However, DC-SIGN, but not CD150, mediates trans-infection of MV to T-lymphocytes independent of DC infection. Together these data suggest a prominent role for DCs during the initiation, dissemination, and clearance of MV infection.

Human Langerhans cells capture measles virus through Langerin and present viral antigens to CD4+ T cells but are incapable of cross-presentation

Langerhans cells (LCs) are a subset of DCs that reside in the upper respiratory tract and are ideally suited to sense respiratory virus infections. Measles virus (MV) is a highly infectious lymphotropic and myelotropic virus that enters the host via the respiratory tract. Here, we show that human primary LCs are capable of capturing MV through the C‐type lectin Langerin. Both immature and mature LCs presented MV‐derived antigens in the context of HLA class II to MV‐specific CD4+ T cells. Immature LCs were not susceptible to productive infection by MV and did not present endogenous viral antigens in the context of HLA class I. In contrast, mature LCs could be infected by MV and presented de novo synthesized viral antigens to MV‐specific CD8+ T cells. Notably, neither immature nor mature LCs were able to cross‐present exogenous UV‐inactivated MV or MV‐infected apoptotic cells. The lack of direct infection of immature LCs, and the inability of both immature and mature LCs to cross‐present MV antigens, suggest that human LCs may not be directly involved in priming MV‐specific CD8+ T cells. Immune activation of LCs seems a prerequisite for MV infection of LCs and subsequent CD8+ T‐cell priming via the endogenous antigen presentation pathway.

Characterization of murine MGL1 and MGL2 C-type lectins: Distinct glycan specificities and tumor binding properties

Antigen presenting cells (APC) express a variety of pattern recognition receptors, including the C-type lectin receptors (CLR) that specifically recognize carbohydrate structures expressed on self-tissue and pathogens. The CLR play an important role in antigen uptake and presentation and have been shown to mediate cellular interactions. The ligand specificity of the human macrophage galactose-type lectin (MGL) has been characterized extensively. Here, we set out to determine the glycan specificity of the murine homologues, MGL1 and MGL2, using a glycan array. Murine MGL1 was found to be highly specific for Lewis X and Lewis A structures, whereas mMGL2, more similar to the human MGL, recognized N-acetylgalactosamine (GalNAc) and galactose, including the O-linked Tn-antigen, TF-antigen and core 2. The generation of MGL1 and MGL2-Fc proteins allowed us to identify ligands in lymph nodes, and MGL1-Fc additionally recognized high endothelial venules. Strikingly, MGL2 interacted strongly to adenocarcinoma cells, suggesting a potential role in tumor immunity.

Interaction of SIGNR1 expressed by marginal zone macrophages with marginal zone B cells is essential to early IgM responses against Streptococcus pneumoniae

The spleen plays a pivotal role in the immune defense against encapsulated bacteria such as Streptococcus pneumoniae. Murine splenic marginal zone macrophages express the C-type lectin SIGNR1, which is crucial for the capture of S. pneumoniae from blood. In this study, we demonstrate that SIGNR1 is able to interact in vitro with the juxtaposing marginal zone B cell population, which is responsible for the production of the early IgM response against the S. pneumoniae-epitope phosphorylcholine. Strikingly, SIGNR1-deficient mice display a reduction in the marginal zone B cell population. In addition, ex vivo B cell stimulation assays demonstrate a decrease in phosphorylcholine specificity in the splenic B cell population derived from SIGNR1-deficient mice, whereas the total IgM response is unaffected. Therefore, we hypothesize that antigens are presented by SIGNR1 expressed by marginal zone macrophages to the developing marginal zone B cell population thereby influencing the repertoire of this B cell population, which is pivotal for the early immune response against encapsulated bacteria such as S. pneumoniae.

Design of neo-glycoconjugates that target the mannose receptor and enhance TLR-independent cross-presentation and Th1 polarization.

Cross‐presentation is an important mechanism by which DCs present exogenous antigens on MHC‐I molecules, and activate CD8+ T cells, cells that are crucial for the elimination of tumors. We investigated the feasibility of exploiting the capacity of the mannose receptor (MR) to improve both cross‐presentation of tumor antigens and Th polarization, processes that are pivotal for the anti‐tumor potency of cytotoxic T cells. To this end, we selected two glycan ligands of the MR, 3‐sulfo‐LewisA and tri‐GlcNAc (N‐acetylglucosamine), to conjugate to the model antigen OVA and assessed in vitro the effect on antigen presentation and Th differentiation. Our results demonstrate that conjugation of either 3‐sulfo‐LewisA or tri‐GlcNAc specifically directs antigen to the MR. Both neo‐glycoconjugates showed, even at low doses, improved uptake as compared with native OVA, resulting in enhanced cross‐presentation. Using MR−/− and MyD88‐TRIFF−/− bone marrow‐derived DCs (BMDCs), we show that the cross‐presentation of the neo‐glycoconjugates is dependent on MR and independent of TLR‐mediated signaling. Whereas proliferation of antigen‐specific CD4+ T cells was unchanged, stimulation with neo‐glycoconjugate‐loaded DCs enhanced the generation of IFN‐γ‐producing T cells. We conclude that modification of antigen with either 3‐sulfo‐LewisA or tri‐GlcNAc enhances cross‐presentation and permits Th1 skewing, through specific targeting of the MR, which may be beneficial for DC‐based vaccination strategies to treat cancer.

Tumour-associated glycan modifications of antigen enhance MGL2 dependent uptake and MHC class I restricted CD8 T cell responses

We recently showed that MGL2 specifically binds tumour‐associated glycan N‐acetylgalactosamine (GalNAc). We here demonstrate that modification of an antigen with tumour‐associated glycan GalNAc, targets antigen specifically to the MGL2 on bone marrow derived (BM)‐DCs and splenic DCs. Glycan‐modification of antigen with GalNAc that mimics tumour‐associated glycosylation, promoted antigen internalisation in DCs and presentation to CD4 T cells, as well as differentiation of IFN‐γ producing CD4 T cells. Furthermore, GalNAc modified antigen enhanced cross‐presentation of both BM‐DCs and primary splenic DCs resulting in enhanced antigen specific CD8 T cell responses. Using MyD88‐TRIFF−/− BM‐DCs we demonstrate that the enhanced cross‐presentation of the GalNAc modified antigen is TLR independent. Our data strongly suggest that tumour‐associated GalNAc modification of antigen targets MGL on DCs and greatly enhances both MHC class II and class I presentation in a TLR independent manner.