Synthetic Abortive HIV-1 RNAs Induce Potent Antiviral Immunity.

Strong innate and adaptive immune responses are paramount in combating viral infections. Dendritic cells (DCs) detect viral infections via cytosolic RIG-I like receptors (RLRs) RIG-I and MDA5 leading to MAVS-induced immunity. The DEAD-box RNA helicase DDX3 senses abortive human immunodeficiency virus 1 (HIV-1) transcripts and induces MAVS-dependent type I interferon (IFN) responses, suggesting that abortive HIV-1 RNA transcripts induce antiviral immunity. Little is known about the induction of antiviral immunity by DDX3-ligand abortive HIV-1 RNA. Here we synthesized a 58 nucleotide-long capped RNA (HIV-1 Cap-RNA58) that mimics abortive HIV-1 RNA transcripts. HIV-1 Cap-RNA58 induced potent type I IFN responses in monocyte-derived DCs, monocytes, macrophages and primary CD1c+ DCs. Compared with RLR agonist poly-I:C, HIV-1 Cap-RNA58 induced comparable levels of type I IFN responses, identifying HIV-1 Cap-RNA58 as a potent trigger of antiviral immunity. In monocyte-derived DCs, HIV-1 Cap-RNA58 activated the transcription factors IRF3 and NF-κB. Moreover, HIV-1 Cap-RNA58 induced DC maturation and the expression of pro-inflammatory cytokines. HIV-1 Cap-RNA58-stimulated DCs induced proliferation of CD4+ and CD8+ T cells and differentiated naïve T helper (TH) cells toward a TH2 phenotype. Importantly, treatment of DCs with HIV-1 Cap-RNA58 resulted in an efficient antiviral innate immune response that reduced ongoing HIV-1 replication in DCs. Our data strongly suggest that HIV-1 Cap-RNA58 induces potent innate and adaptive immune responses, making it an interesting addition in vaccine design strategies.

DDX3 in HIV-1 infection and sensing: A paradox.

HIV-1 sensors and their signaling features have been an ongoing topic of intense research over the last decade, as these mechanisms fail to establish protective immunity against HIV-1. Here, we discuss how HIV-1 infects dendritic cells (DCs) and which sensors play a role in recognizing viral DNA and RNA in these specialized immune cells. We will elaborate on the RNA helicase DDX3, which is crucial in translation initiation of HIV-1 mRNA, but also fulfills an important role as RNA sensor and inducer of antiviral immunity in DCs. As DDX3 is indispensable for HIV-1 replication, the virus cannot escape sensing by DDX3, which is an important aspect of its function. Last but not least, we will discuss how HIV-1 suppresses DDX3 sensing and how this impacts the viral load in HIV-1-infected individuals.

RIG-I-like receptor activation by dengue virus drives follicular T helper cell formation and antibody production.

Follicular T helper cells (TFH) are fundamental in orchestrating effective antibody-mediated responses critical for immunity against viral infections and effective vaccines. However, it is unclear how virus infection leads to TFH induction. We here show that dengue virus (DENV) infection of human dendritic cells (DCs) drives TFH formation via crosstalk of RIG-I-like receptor (RLR) RIG-I and MDA5 with type I Interferon (IFN) signaling. DENV infection leads to RLR-dependent IKKε activation, which phosphorylates IFNα/ß receptor-induced STAT1 to drive IL-27 production via the transcriptional complex ISGF3. Inhibiting RLR activation as well as neutralizing antibodies against IL-27 prevented TFH formation. DENV-induced CXCR5+PD-1+Bcl-6+ TFH cells secreted IL-21 and activated B cells to produce IgM and IgG. Notably, RLR activation by synthetic ligands also induced IL-27 secretion and TFH polarization. These results identify an innate mechanism by which antibodies develop during viral disease and identify RLR ligands as potent adjuvants for TFH-promoting vaccination strategies.

RIG-I-like Receptor Triggering by Dengue Virus Drives Dendritic Cell Immune Activation and TH1 Differentiation.

Dengue virus (DENV) causes 400 million infections annually and is one of several viruses that can cause viral hemorrhagic fever, which is characterized by uncontrolled immune activation resulting in high fever and internal bleeding. Although the underlying mechanisms are unknown, massive cytokine secretion is thought to be involved. Dendritic cells (DCs) are the main target cells of DENV, and we investigated their role in DENV-induced cytokine production and adaptive immune responses. DENV infection induced DC maturation and secretion of IL-1ß, IL-6, and TNF. Inhibition of DENV RNA replication abrogated these responses. Notably, silencing of RNA sensors RIG-I or MDA5 abrogated DC maturation, as well as cytokine responses by DENV-infected DCs. DC maturation was induced by type I IFN responses because inhibition of IFN-α/ß receptor signaling abrogated DENV-induced DC maturation. Moreover, DENV infection of DCs resulted in CCL2, CCL3, and CCL4 expression, which was abrogated after RIG-I and MDA5 silencing. DCs play an essential role in TH cell differentiation, and we show that RIG-I and MDA5 triggering by DENV leads to TH1 polarization, which is characterized by high levels of IFN-γ. Notably, cytokines IL-6, TNF, and IFN-γ and chemokines CCL2, CCL3, and CCL4 have been associated with disease severity, endothelial dysfunction, and vasodilation. Therefore, we identified RIG-I and MDA5 as critical players in innate and adaptive immune responses against DENV, and targeting these receptors has the potential to decrease hemorrhagic fever in patients.

Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants.

Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of some helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct the helminth glycoproteins omega-1 and kappa-5, two major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein's Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens.

HIV-1 blocks the signaling adaptor MAVS to evade antiviral host defense after sensing of abortive HIV-1 RNA by the host helicase DDX3.

The mechanisms by which human immunodeficiency virus 1 (HIV-1) avoids immune surveillance by dendritic cells (DCs), and thereby prevents protective adaptive immune responses, remain poorly understood. Here we showed that HIV-1 actively arrested antiviral immune responses by DCs, which contributed to efficient HIV-1 replication in infected individuals. We identified the RNA helicase DDX3 as an HIV-1 sensor that bound abortive HIV-1 RNA after HIV-1 infection and induced DC maturation and type I interferon responses via the signaling adaptor MAVS. Notably, HIV-1 recognition by the C-type lectin receptor DC-SIGN activated the mitotic kinase PLK1, which suppressed signaling downstream of MAVS, thereby interfering with intrinsic host defense during HIV-1 infection. Finally, we showed that PLK1-mediated suppression of DDX3-MAVS signaling was a viral strategy that accelerated HIV-1 replication in infected individuals.

C-type lectin receptors in the control of T helper cell differentiation.

Pathogen recognition by C-type lectin receptors (CLRs) expressed by dendritic cells is important not only for antigen presentation, but also for the induction of appropriate adaptive immune responses via T helper (TH) cell differentiation. CLRs act either by themselves or in cooperation with other receptors, such as other CLRs, Toll-like receptors and interferon receptors, to induce signalling pathways that trigger specialized cytokine programmes for polarization of TH cell differentiation. In this Review, we discuss how triggering of the prototypical CLRs leads to distinct pathogen-tailored TH cell responses and how we can harness our expanding knowledge for vaccine design and the treatment of inflammatory and malignant diseases.

Fucose-based PAMPs prime dendritic cells for follicular T helper cell polarization via DC-SIGN-dependent IL-27 production.

Dendritic cells (DCs) orchestrate antibody-mediated responses to combat extracellular pathogens including parasites by initiating T helper cell differentiation. Here we demonstrate that carbohydrate-specific signalling by DC-SIGN drives follicular T helper cell (TFH) differentiation via IL-27 expression. Fucose, but not mannose, engagement of DC-SIGN results in activation of IKKε, which collaborates with type I IFNR signalling to induce formation and activation of transcription factor ISGF3. Notably, ISGF3 induces expression of IL-27 subunit p28, and subsequent IL-27 secreted by DC-SIGN-primed DCs is pivotal for the induction of Bcl-6(+)CXCR5(+)PD-1(hi)Foxp1(lo) TFH cells, IL-21 secretion by TFH cells and T-cell-dependent IgG production by B cells. Thus, we have identified an essential role for DC-SIGN-induced ISGF3 by fucose-based PAMPs in driving IL-27 and subsequent TFH polarization, which might be harnessed for vaccination design.

Antagonism of the phosphatase PP1 by the measles virus V protein is required for innate immune escape of MDA5.

The cytosolic sensor MDA5 is crucial for antiviral innate immune defense against various RNA viruses including measles virus; as such, many viruses have evolved strategies to antagonize the antiviral activity of MDA5. Here, we show that measles virus escapes MDA5 detection by targeting the phosphatases PP1α and PP1γ, which regulate MDA5 activity by removing an inhibitory phosphorylation mark. The V proteins of measles virus and the related paramyxovirus Nipah virus interact with PP1α/γ, preventing PP1-mediated dephosphorylation of MDA5 and thereby its activation. The PP1 interaction with the measles V protein is mediated by a conserved PP1-binding motif in the C-terminal region of the V protein. A recombinant measles virus expressing a mutant V protein deficient in PP1 binding is unable to antagonize MDA5 and is growth impaired due to its inability to suppress interferon induction. This identifies PP1 antagonism as a mechanism employed by paramyxoviruses for evading innate immune recognition.

Measles virus suppresses RIG-I-like receptor activation in dendritic cells via DC-SIGN-mediated inhibition of PP1 phosphatases.

Dendritic cells (DCs) are targets of measles virus (MV) and play central roles in viral dissemination. However, DCs express the RIG-I-like receptors (RLRs) RIG-I and Mda5 that sense MV and induce type I interferon (IFN) production. Given the potency of this antiviral response, RLRs are tightly regulated at various steps, including dephosphorylation by PP1 phosphatases, which induces their activation. We demonstrate that MV suppresses RIG-I and Mda5 by activating the C-type lectin DC-SIGN and inducing signaling that prevents RLR dephosphorylation. MV binding to DC-SIGN leads to activation of the kinase Raf-1, which induces the association of PP1 inhibitor I-1 with GADD34-PP1 holoenzymes, thereby inhibiting phosphatase activity. Consequently, GADD34-PP1 holoenzymes are unable to dephosphorylate RIG-I and Mda5, hence suppressing type I IFN responses and enhancing MV replication. Blocking DC-SIGN signaling allows RLR activation and suppresses MV infection of DCs. Thus, MV subverts DC-SIGN to control RLR activation and escape antiviral responses.

Fucose-specific DC-SIGN signalling directs T helper cell type-2 responses via IKKε- and CYLD-dependent Bcl3 activation.

Carbohydrate-specific signalling through DC-SIGN provides dendritic cells with plasticity to tailor immunity to the nature of invading microbes. Here we demonstrate that recognition of fucose-expressing extracellular pathogens like Schistosoma mansoni and Helicobacter pylori by DC-SIGN favors T helper cell type-2 (TH2) responses via activation of atypical NF-κB family member Bcl3. Crosstalk between TLR and DC-SIGN signalling results in TLR-induced MK2-mediated phosphorylation of LSP1, associated with DC-SIGN, upon fucose binding. Subsequently, IKKε and CYLD are recruited to phosphorylated LSP1. IKKε activation is pivotal for suppression of CYLD deubiquitinase activity and subsequent nuclear translocation of ubiquitinated Bcl3. Bcl3 activation represses TLR-induced proinflammatory cytokine expression, while enhancing interleukin-10 (IL-10) and TH2-attracting chemokine expression, shifting TH differentiation from TH1 to TH2 polarization. Thus, DC-SIGN directs adaptive TH2 immunity to fucose-expressing pathogens via an IKKε-CYLD-dependent signalling pathway leading to Bcl3 activation, which might be targeted in vaccination strategies or to prevent aberrant inflammation and allergy.

Fungal engagement of the C-type lectin mincle suppresses dectin-1-induced antifungal immunity.

Recognition of fungal pathogens by C-type lectin receptor (CLR) dectin-1 on human dendritic cells is essential for triggering protective antifungal TH1 and TH17 immune responses. We show that Fonsecaea monophora, a causative agent of chromoblastomycosis, a chronic fungal skin infection, evades these antifungal responses by engaging CLR mincle and suppressing IL-12, which drives TH1 differentiation. Dectin-1 triggering by F. monophora activates transcription factor IRF1, which is crucial for IL12A transcription via nucleosome remodeling. However, simultaneous F. monophora binding to mincle induces an E3 ubiquitin ligase Mdm2-dependent degradation pathway, via Syk-CARD9-mediated PKB signaling, that leads to loss of nuclear IRF1 activity, hence blocking IL12A transcription. The absence of IL-12 leads to impaired TH1 responses and promotes TH2 polarization. Notably, mincle is similarly exploited by other chromoblastomycosis-associated fungi to redirect TH responses. Thus, mincle is a fungal receptor that can suppress antifungal immunity and, as such, is a potential therapeutic target.

Selective C-Rel activation via Malt1 controls anti-fungal T(H)-17 immunity by dectin-1 and dectin-2.

C-type lectins dectin-1 and dectin-2 on dendritic cells elicit protective immunity against fungal infections through induction of T(H)1 and T(H)-17 cellular responses. Fungal recognition by dectin-1 on human dendritic cells engages the CARD9-Bcl10-Malt1 module to activate NF-κB. Here we demonstrate that Malt1 recruitment is pivotal to T(H)-17 immunity by selective activation of NF-κB subunit c-Rel, which induces expression of T(H)-17-polarizing cytokines IL-1ß and IL-23p19. Malt1 inhibition abrogates c-Rel activation and T(H)-17 immunity to Candida species. We found that Malt1-mediated activation of c-Rel is similarly essential to induction of T(H)-17-polarizing cytokines by dectin-2. Whereas dectin-1 activates all NF-κB subunits, dectin-2 selectively activates c-Rel, signifying a specialized T(H)-17-enhancing function for dectin-2 in anti-fungal immunity by human dendritic cells. Thus, dectin-1 and dectin-2 control adaptive T(H)-17 immunity to fungi via Malt1-dependent activation of c-Rel.

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-kappaB 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-kappaB 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.

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.

Dectin-1 directs T helper cell differentiation by controlling noncanonical NF-kappaB activation through Raf-1 and Syk.

The C-type lectin dectin-1 activates the transcription factor NF-kappaB through a Syk kinase-dependent signaling pathway to induce antifungal immunity. Here we show that dectin-1 expressed on human dendritic cells activates not only the Syk-dependent canonical NF-kappaB subunits p65 and c-Rel, but also the noncanonical NF-kappaB subunit RelB. Dectin-1, when stimulated by the beta-glucan curdlan or by Candida albicans, induced a second signaling pathway mediated by the serine-threonine kinase Raf-1, which integrated with the Syk pathway at the point of NF-kappaB activation. Raf-1 antagonized Syk-induced RelB activation by promoting sequestration of RelB into inactive p65-RelB dimers, thereby altering T helper cell differentiation. Thus, dectin-1 activates two independent signaling pathways, one through Syk and one through Raf-1, to induce immune responses.

Innate signaling by the C-type lectin DC-SIGN dictates immune responses.

Effective immune responses depend on the recognition of pathogens by dendritic cells (DCs) through pattern recognition receptors (PRRs). These receptors induce specific signaling pathways that lead to the induction of immune responses against the pathogens. It is becoming evident that C-type lectins are also important PRRs. In particular, the C-type lectin DC-SIGN has emerged as a key player in the induction of immune responses against numerous pathogens by modulating TLR-induced activation. Recent reports have begun to elucidate the molecular mechanisms underlying these immune responses. Upon pathogen binding, DC-SIGN induces an intracellular signaling pathway with a central role for the serine/threonine kinase Raf-1. For several pathogens that interact with DC-SIGN, including Mycobacterium tuberculosis and HIV-1, Raf-1 activation leads to acetylation of NF-kappaB subunit p65, which induces specific gene transcription profiles. In addition, other DC-SIGN-ligands induce different signaling pathways downstream of Raf-1, indicating that DC-SIGN-signaling is tailored to the pathogen. In this review we will discuss in detail the current knowledge about DC-SIGN signaling and its implications on immunity.

TNF-alpha and TLR agonists increase susceptibility to HIV-1 transmission by human Langerhans cells ex vivo.

Genital coinfections increase an individual's risk of becoming infected with HIV-1 by sexual contact. Several mechanisms have been proposed to explain this, such as the presence of ulceration and bleeding caused by the coinfecting pathogen. Here we demonstrate that Langerhans cells (LCs) are involved in the increased susceptibility to HIV-1 in the presence of genital coinfections. Although LCs are a target for HIV-1 infection in genital tissues, we found that immature LCs did not efficiently mediate HIV-1 transmission in an ex vivo human skin explant model. However, the inflammatory stimuli TNF-alpha and Pam3CysSerLys4 (Pam3CSK4), the ligand for the TLR1/TLR2 heterodimer, strongly increased HIV-1 transmission by LCs through distinct mechanisms. TNF-alpha enhanced transmission by increasing HIV-1 replication in LCs, whereas Pam3CSK4 acted by increasing LC capture of HIV-1 and subsequent trans-infection of T cells. Genital infections such as Candida albicans and Neisseria gonorrhea not only triggered TLRs but also induced TNF-alpha production in vaginal and skin explants. Thus, during coinfection, LCs could be directly activated by pathogenic structures and indirectly activated by inflammatory factors, thereby increasing the risk of acquiring HIV-1. Our data demonstrate a decisive role for LCs in HIV-1 transmission during genital coinfections and suggest antiinflammatory therapies as potential strategies to prevent HIV-1 transmission.

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-alpha 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.

DC-SIGN mediates adhesion and rolling of dendritic cells on primary human umbilical vein endothelial cells through LewisY antigen expressed on ICAM-2.

Immature dendritic cells (DCs) are recruited from blood into tissues to patrol for foreign antigens. After antigen uptake and processing, DCs mature and migrate to the secondary lymphoid organs where they initiate immune responses. DC-SIGN is a DC-specific C-type lectin that acts both as a pattern recognition receptor and as an adhesion molecule. As an adhesion molecule, DC-SIGN is able to mediate rolling and adhesion over endothelial cells under shear flow. In this study, we show that the binding partner of DC-SIGN on endothelial cells is the glycan epitope Lewis(Y) (Le(Y)), expressed on ICAM-2. The interaction between DC-SIGN on dendritic cells and ICAM-2 on endothelial cells is strictly glycan-specific. ICAM-2 expressed on CHO cells only served as a ligand for DC-SIGN when properly glycosylated, underscoring its function as a scaffolding protein. The expression of Le(Y) in endothelial cells is directed by the enzyme FUT1. Silencing of FUT1 results in a decrease in the rolling and adhesion of immature DCs over endothelial cells. The identification of Le(Y) as the carbohydrate ligand of DC-SIGN in endothelial cells opens new possibilities for the manipulation of DC migration.