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The non-pathogenic TH17 subset of helper T cells clears fungal infections, whereas pathogenic TH17 cells cause inflammation and tissue damage; however, the mechanisms controlling these distinct responses remain unclear. Here we found that fungi sensing by the C-type lectin dectin-1 in human dendritic cells (DCs) directed the polarization of non-pathogenic TH17 cells. Dectin-1 signaling triggered transient and intermediate expression of interferon (IFN)-β in DCs, which was mediated by the opposed activities of transcription factors IRF1 and IRF5. IFN-β-induced signaling led to integrin αvβ8 expression directly and to the release of the active form of the cytokine transforming growth factor (TGF)-β indirectly. Uncontrolled IFN-β responses as a result of IRF1 deficiency induced high expression of the IFN-stimulated gene BST2 in DCs and restrained TGF-β activation. Active TGF-β was required for polarization of non-pathogenic TH17 cells, whereas pathogenic TH17 cells developed in the absence of active TGF-β. Thus, dectin-1-mediated modulation of type I IFN responses allowed TGF-β activation and non-pathogenic TH17 cell development during fungal infections in humans.

Pathogens trigger multiple pattern recognition receptors (PRRs) that together dictate innate and adaptive immune responses. Understanding the crosstalk between PRRs is important to enhance vaccine efficacy. Abortive HIV-1 RNA transcripts are produced during acute and chronic HIV-1 infection and are known ligands for different PRRs, leading to antiviral and proinflammatory responses. Here, we have investigated the crosstalk between responses induced by these 58 nucleotide-long HIV-1 RNA transcripts and different TLR ligands. Costimulation of dendritic cells (DCs) with abortive HIV-1 RNA and TLR7/8 agonist R848, but not other TLR agonists, resulted in enhanced antiviral type I IFN responses as well as adaptive immune responses via the induction of DC-mediated T helper 1 (TH1) responses and IFNγ+CD8+ T cells. Our data underscore the importance of crosstalk between abortive HIV-1 RNA and R848-induced signaling for the induction of effective antiviral immunity.

The non-pathogenic T

The mitochondrial antiviral protein MAVS is a key player in the induction of antiviral responses, however, human immunodeficiency virus 1 (HIV-1) is able to suppress these responses. Two linked single nucleotide polymorphisms (SNPs) in the MAVS gene render MAVS insensitive to HIV-1-dependent suppression, and have been shown to be associated with a lower viral load at set point and delayed increase of viral load during disease progression. Here, we studied the underlying mechanisms involved in the control of viral replication in individuals homozygous for this MAVS genotype. We observed that individuals with the MAVS minor genotype had more stable total CD4+ T cell counts during a 7-year follow up and had lower cell-associated proviral DNA loads. Genetic variation in MAVS did not affect immune activation levels, however, a significantly lower percentage of na&iuml, ve CD4+ but not CD8+ T cells was observed in the MAVS minor genotype. In vitro HIV-1 infection of peripheral blood mononuclear cells (PBMCs) from healthy donors with the MAVS minor genotype resulted in decreased viral replication. Although the precise underlying mechanism remains unclear, our data suggest that the protective effect of the MAVS minor genotype may be exerted by the initiation of local innate responses affecting viral replication and CD4+ T cell susceptibility.

Here, the authors discuss the role of C-type lectin receptor signalling pathways in the control of T helper cell differentiation and examine how these receptors and pathways can be harnessed for vaccine strategies. 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.

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.

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.

SummaryRecognition 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.

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-1β, 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/β 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.

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/β 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., Author summary Strong antibody production is critical for effective immune responses against viral infections and is a primary factor in the development of successful vaccines. However, it is unclear how virus infection leads to effective antibody responses. Dengue virus (DENV) is known to induce potent antibody production, although the underlying mechanism is poorly understood. Dendritic cells (DCs) are professional sentinels of the immune system and crucial for induction of immune responses. Here we show that DENV infection of human DCs leads to robust antibody production by inducing a specific T helper cell type (also called follicular T helper or TFH) that specializes in stimulating antibody production by B cells. Our data show that DENV replication triggers a viral detection system consisting of sensors RIG-I and MDA5, which specifically induce factors such as IL-27 that are essential for TFH induction. Our data demonstrate that this viral detection system is especially powerful to induce antibody production. Indeed, synthetic molecules that trigger this viral detection mechanism induced superior antibody production compared to other activation signals. Thus, we have identified a viral detection mechanism that leads to strong antibody production and its importance in DENV infection as well as its potential in vaccinations.

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-1β, 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/β 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 T

Host-pathogen interactions have coevolved for many years. On the one hand, the human immune system consists of innate and adaptive immune cells that function to defeat pathogens, and on the other hand, pathogens have coevolved to use the system for their own propagation. C-type lectins are conserved receptors recognizing carbohydrate structures on viruses, bacteria, parasites, and fungi. C-type lectins such as DC-SIGN, langerin, and dectin-1 are expressed by dendritic cell subsets and macrophages. Pathogen recognition by C-type lectins triggers signaling pathways that lead to the expression of specific cytokines which subsequently instruct adaptive T helper immune responses. T helper cell differentiation is crucial for initiating proper adaptive immune responses; some pathogens, however, use pattern recognition receptors like C-type lectins to subvert immune responses for survival. This review provides an update on the role of C-type lectins in HIV-1, mycobacterial, and Candida infections, and the coevolution of hosts and pathogens.

Production of the proinflammatory cytokine interleukin 1 beta (IL-1 beta) by dendritic cells is crucial in host defense. Here we identify a previously unknown role for dectin-1 in the activation of a noncanonical caspase-8 inflammasome in response to fungi and mycobacteria. Dectin-1 induced both the production and maturation of IL-1 beta through signaling routes mediated by the kinase Syk. Whereas the CARD9-Bcl-10-MALT1 scaffold directed IL1B transcription, the recruitment of MALT1-caspase-8 and ASC into this scaffold was crucial for processing of pro-IL-1 beta by caspase-8. In contrast to activation of the canonical caspase-1 inflammasome, which requires additional activation of cytosolic receptors, activation of the noncanonical caspase-8 inflammasome was independent of pathogen internalization. Thus, dectin-1 acted as an extracellular sensor for pathogens that induced both IL-1 beta production and maturation through a noncanonical caspase-8-dependent inflammasome for protective immunity

Key Points Crosstalk between pattern recognition receptors (PRRs) expressed by dendritic cells orchestrates T helper (TH) cell differentiation through the induction of specific cytokine expression profiles, tailored to invading pathogens. C-type lectin receptors (CLRs) have an important role in orchestrating the induction of signalling pathways that regulate adaptive immune responses.CLRs can control adaptive immunity at various levels by inducing signalling on their own, through crosstalk with other PRRs or by inducing carbohydrate-specific signalling pathways.DC-specific ICAM3-grabbing non-integrin (DC-SIGN) interacts with mannose-carrying pathogens including Mycobacterium tuberculosis, HIV-1, measles virus and Candida albicans to activate the serine/threonine protein kinase RAF1. RAF1 signalling leads to the acetylation of Toll-like receptor (TLR)-activated nuclear factor-κB (NF-κB) subunit p65 and affects cytokine expression, such as inducing the upregulation of interleukin-10 (IL-10).DC-associated C-type lectin 1 (dectin 1) triggering by a broad range of fungal pathogens, such as C. albicans, Aspergillus fumigatus and Pneumocystis carinii, results in protective antifungal immunity through the crosstalk of two independent signalling pathways — one through spleen tyrosine kinase (SYK) and one through RAF1 — that are essential for the expression of TH1 and TH17 cell polarizing cytokines.Crosstalk between the SYK and RAF1 pathways is both synergistic and antagonizing to fine-tune NF-κB activity: although Ser276 phosphorylation of p65 leads to enhanced transcriptional activity of p65 itself through acetylation, it also inhibits the transcriptional activity of the NF-κB subunit RELB by sequestering it in p65–RELB dimers, which are transcriptionally inactive.The diversity in CLR-mediated signalling provides some major challenges for the researches to elucidate and manipulate the signalling properties of this exciting family of receptors. However, the recent advances strongly support the use of CLR targeting vaccination strategies using dendritic cells to induce or redirect adaptive immune responses as well as improve antigen delivery., Here, Teunis Geijtenbeek and Sonja Gringhuis discuss the role of the signalling pathways induced by C-type lectin receptors in determining T helper cell lineage commitment and describe how these pathways can be exploited for the development of new vaccination strategies., C-type lectin receptors (CLRs) expressed by dendritic cells are crucial for tailoring immune responses to pathogens. Following pathogen binding, CLRs trigger distinct signalling pathways that induce the expression of specific cytokines which determine T cell polarization fates. Some CLRs can induce signalling pathways that directly activate nuclear factor-κB, whereas other CLRs affect signalling by Toll-like receptors. Dissecting these signalling pathways and their effects on host immune cells is essential to understand the molecular mechanisms involved in the induction of adaptive immune responses. In this Review we describe the role of CLR signalling in regulating adaptive immunity and immunopathogenesis and discuss how this knowledge can be harnessed for the development of innovative vaccination approaches.

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.

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.

Cytokine secretion profiles of activated T cells are critical for maintaining the immunologic balance between protection and tolerance. In mice, several cytokines have been reported to exhibit monoallelic expression. Previously, we found that the human interleukin-1 alpha (IL1A) gene exhibits a stable allele-specific expression pattern in CD4+ T-cell clones. We investigated whether DNA methylation is involved in the allele-specific expression of IL-1alpha. Here, we show that differential methylation of CpGs in the proximal promoter region is associated with allele-specific expression of IL-1alpha in CD4+ T cells. The differential methylation pattern is already observed in naive T cells. In keratinocytes, which constitutively produce IL-1alpha, the proximal promoter is hypomethylated. CpGs located further upstream and in intron 4 were almost all methylated, irrespective of expression. Treatment of nonexpressing cells and of T-cell clones with 5-aza-2'deoxycytidine induced IL-1alpha expression in the nonexpressing cells and induced expression of the formerly silent allele in T-cell clones. In addition, electrophoretic mobility shift assays showed that methylation of CpGs in the proximal promoter resulted in direct inhibition of binding of nuclear factor(s). Taken together, these results suggest that allele-specific expression of IL-1alpha in CD4+ cells is achieved, at least in part, by differential methylation of the promoter.

The endothelium plays a central role in the logistics of the immune system by allowing the selective transmigration of leukocytes, as well as the maintenance of the circulation and coagulation homeostasis. Evidence is increasing that the carbohydrate composition of the endothelial cell surface is critical for the cells to exert their physiological function. The major aim of this study is to unravel the mechanisms underlying the expression of carbohydrate structures by endothelial cells, which are involved in leukocyte adhesion and migration. Using quantitative real-time PCR, the expression profile of a selected group of 74 glycosylation-related genes has been determined in human umbilical vein endothelial cells (HUVEC) and human foreskin microvascular endothelial cells (FMVEC) under control and TNFα-induced conditions. The set of genes comprised 59 glycosyltransferases, 6 mannosidases and 9 sulfotransferases. In parallel, the overall cell surface glycan profile has been assessed by the use of glycan-specific lectins and monoclonal antibodies. The results demonstrate that HUVEC and FMVEC differ substantially in the expression of glycosylation-related genes and, accordingly, also in the presence of different glycan epitopes on the cell membrane. Induction of an inflamed phenotype of the cells by treatment with TNFα differentially modulates a set of these genes in HUVEC and FMVEC resulting in a change in the cell membrane associated glycans that are of importance in inflammation-related endothelial cell-surface processes. © 2005 Wiley-Liss, Inc.

Posttranscriptional regulation of gene expression offers a powerful mechanism to swiftly respond to extracellular stimuli and either up- or downregulate the expression of targeted proteins. An important posttranscriptional mechanism exerts its effects on mRNA stability. mRNA molecules contain specific cis-acting elements that are bound by trans-acting factors that affect the rate at which mRNA is degraded. The most common cis-acting elements in mammalian cells involved in rapid mRNA decay are the AU-rich elements (ARE). AREs contain one or more repeats of the pentanucleotide AUUUA sequence within the 3′ untranslated regions (3′UTR) of short-lived mRNA molecules. The regulation of gene expression through ARE-mediated modulation of mRNA plays a critical role during cell growth and differentation, in apoptosis, and in the immune response (2, 8, 34). Various ARE-binding proteins (AUBPs) have been previously described. Both destabilizing and stabilizing factors have been identified which dynamically modulate mRNA turnover in an opposing fashion. Although the exact mechanisms by which the RNA/protein interactions affect the mRNA deadenylation and decay remain elusive, it has been reported that the destabilizing AUBPs directly or indirectly recruit the exosome, a multiprotein complex of 3′-to-5′ exoribonucleases, promoting mRNA decay (7, 15). Proteins belonging to the class of destabilizing AUBPs are AU-rich RNA-binding factor 1 (AUF1), also known as heterogeneous nuclear ribonucleoprotein D; K homology splicing regulatory protein (KSRP); tristetraprolin (TTP); and the TTP-relative butyrate response factor-1 (BRF1) (3, 15, 32, 37). Proteins that have been shown to increase the stability of mRNA include Hu antigen R, nuclear factor 90, and nucleolin (4, 34, 36). These factors are thought to block mRNA decay by preventing binding of the destabilizing AUBPs and thus the recruitment of the exosome. Another layer of complexity is added by AUBPs like CUGBP2, which facilitate mRNA stabilization but at the same time inhibit translation (23). Despite a large knowledge base about the AUBPs involved in regulating mRNA stability, how mRNA binds the factors and how the switch between destabilizing and stabilizing proteins is regulated remain ill defined. A variety of signaling pathways induced by inflammatory stimuli have been implicated in either stabilization or destabilization of mRNAs. Whereas the use of inhibitors and dominant negative mutants of signaling proteins has shown the involvement of TAK1, mitogen-activated protein (MAP) kinase kinase kinase 1, MAP kinase kinase 6, p38, p38 MAP kinase-activated protein kinase 2 (MK2; also known as MAPKAP2), c-jun N-terminal kinase, and extracellular signal-regulated kinase in modulating mRNA turnover, it is unclear how these mechanisms are linked (10, 11, 17, 22, 45). More specifically, MK2 has been shown to be involved in the phosphorylation of the destabilizing factor TTP, influencing the association of TTP with the chaperone protein 14-3-3, yet it remains unresolved how phosphorylation affects the mRNA binding and activity of TTP (9). In contrast, the phosphorylation of AUF1 has been shown to remodel local RNA structures and most likely regulates mRNA turnover by altering the recruitment of the degradation machinery; however, the signaling pathways and kinases responsible for AUF1 phosphorylation in vivo remain elusive (43, 44). Recently, we reported that the stability of the β1,4-galactosyltransferase I (β4GalT1) mRNA is solely mediated through the second AU-rich element (AU2), which in resting primary human umbilical vein endothelial cells (HUVECs) is bound by a destabilizing factor. The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) upregulates the expression of β4GalT1 via mRNA stabilization (13). To further understand how mRNA stability is modulated in response to extracellular stimuli, we investigated the signaling pathways and regulatory mechanisms involved in TNF-α-induced stabilization of β4GalT1 mRNA. It was found that in resting HUVECs, AU2 was bound by a destabilizing complex of TTP and 14-3-3β, resulting in rapid mRNA turnover. We observed that TNF-α induced two distinct signaling pathways, one mediated by inhibitor κB kinase β (IKKβ) and the other was mediated by protein kinase Cδ (PKCδ), which resulted in the dislodgment of the TTP/14-3-3β complex from AU2 and the nuclear translocation of TTP, paralleled by an increase in the β4GalT1 mRNA half-life. We provide a mechanism for these observations through the phosphorylation of 14-3-3β by IKKβ and PKCδ on serine residues Ser132 and Ser60, respectively, which interferes with its binding to TTP and hence the retention of TTP in the cytoplasm.

During the course of an inflammatory response, the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFalpha) triggers endothelial cells to increase the expression levels of adhesion molecules that are pivotal for the rolling, adhesion, and transmigration of leukocytes over the endothelial cell wall. Here we show that TNFalpha, in addition, has a regulatory function in the biosynthesis of proper carbohydrate molecules on endothelial cells that constitute ligands for adhesion molecules on leukocytes. Our data show that TNFalpha induced an increase in the expression of beta1,4-galactosyltransferase-1 (beta4GalT-1) in primary human umbilical vein endothelial cells in a time- and concentration-dependent manner. The beta4GalT-1 mRNA up-regulation correlated with an increase in the Golgi expression and catalytic activity of the enzyme. Furthermore, an enhanced incorporation of galactose was observed in newly synthesized glycoproteins. Analysis of the molecular mechanism behind the up-regulation of beta4GalT-1 showed that the increase in mRNA levels is due to an enhanced stability of the transcripts. These data strongly demonstrate that TNFalpha modulates the glycosylation of endothelial cells by a mechanism that directly enhances the stability of beta4GalT-1 mRNA transcripts.

Transient production of reactive oxygen species (ROS) plays an important role in optimizing transcriptional and proliferative responses to TCR signaling in T lymphocytes. Conversely, chronic oxidative stress leads to decreased proliferative responses and enhanced transcription of inflammatory gene products, and is thought to underlie the altered pathogenic behavior of T lymphocytes in some human diseases, such as rheumatoid arthritis (RA). Although the signaling mechanisms regulating ROS production in T lymphocytes has not been identified, activation of the small GTPase Ras has been shown to couple agonist stimulation to ROS production in other cell types. We find that Ras signaling via Ral stimulates ROS production in human T lymphocytes, and is required for TCR and phorbol ester-induced ROS production. The related small GTPase Rap1 suppresses agonist, Ras and Ral–dependent ROS production through a PI3K–dependent pathway, identifying a novel mechanism by which Rap1 can distally antagonize Ras signaling pathways. In synovial fluid T lymphocytes from RA patients we observed a high rate of endogenous ROS production, correlating with constitutive Ras activation and inhibition of Rap1 activation. Introduction of dominant-negative Ras into synovial fluid T cells restored redox balance, providing evidence that deregulated Ras and Rap1 signaling underlies oxidative stress and consequent altered T cell function observed in RA.

Antibodies bound to pathogens that invade cells are sensed in the cytosol by the receptor TRIM21, which triggers innate signaling that leads to protective immunity.

A hallmark of HIV-1 infection is the lack of sterilizing immunity. Dendritic cells (DCs) are crucial in the induction of immunity, and lack of DC activation might underlie the absence of an effective anti–HIV-1 response. We have investigated how HIV-1 infection affects maturation of DCs. Our data show that even though DCs are productively infected by HIV-1, infection does not induce DC maturation. HIV-1 infection actively suppresses DC maturation, as HIV-1 infection inhibited TLR-induced maturation of DCs and thereby decreased the immune stimulatory capacity of DCs. Interfering with SAMHD1 restriction further increased infection of DCs, but did not lead to DC maturation. Notably, higher infection observed with SAMHD1 depletion correlated with a stronger suppression of maturation. Furthermore, blocking reverse transcription rescued TLR-induced maturation. These data strongly indicate that HIV-1 replication does not trigger immune activation in DCs, but that HIV-1 escapes immune surveillance by actively suppressing DC maturation independent of SAMHD1. Elucidation of the mechanism of suppression can lead to promising targets for therapy or vaccine design.

The cellular response to an extracellular signal starts with the induction of a signaling cascade that transmits the signal through the cytoplasm to the nucleus, resulting in the activation of transcription factors that activate specific target genes. The signaling cascade involves a series of biochemical modifications that include phosphorylation events on tyrosine residues due to the activation of specific protein kinases. Recently, evidence accumulated that reactive oxygen species, including hydrogen peroxide, superoxide, and the hydroxyl radical, are important chemical mediators that regulate the transduction of signals from the membrane to the nucleus by modulating the protein activity by oxidation and reduction. In this report, the redox regulation of signaling involving protein tyrosine kinase activity, in particular in T- and B-lymphocyte signaling, is reviewed.

The integral membrane protein linker for activation of T cells (LAT) is a central adapter protein in the T-cell receptor (TCR)-mediated signaling pathways. The cellular localization of LAT is extremely sensitive to intracellular redox balance alterations. Reduced intracellular levels of the antioxidant glutathione (GSH), a hallmark of chronic oxidative stress, resulted in the membrane displacement of LAT, abrogated TCR-mediated signaling and consequently hyporesponsiveness of T lymphocytes. The membrane displacement of LAT is accompanied by a considerable difference in the mobility of LAT upon native and nonreducing denaturing polyacrylamide gel electrophoresis analysis, a finding indicative of a conformational change. Targeted mutation of redox-sensitive cysteine residues within LAT created LAT mutants which remain membrane anchored under conditions of chronic oxidative stress. The expression of redox-insensitive LAT mutants allows for restoration of TCR-mediated signal transduction, whereas CD28-mediated signaling pathways remained impaired. These results are indicative that the membrane displacement of LAT as a result of redox balance alterations is a consequence of a conformational change interfering with the insertion of LAT into the plasma membrane. Conclusively, the data suggest a role for LAT as a crucial intermediate in the sensitivity of TCR signaling and hence T lymphocytes toward chronic oxidative stress.

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 PP1 alpha and PP1 gamma, which regulate MDA5 activity by removing an inhibitory phosphorylation mark. The V proteins of measles virus and the related paramyxovirus Nipah virus interact with PP1 alpha/gamma, 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

The T lymphocytes that reside in the synovium of the inflamed joints in patients with rheumatoid arthritis display severe hyporesponsiveness upon antigenic stimulation, which is probably due to their constant subjection to high levels of oxidative stress. Here we report that the synovial fluid T lymphocytes exert severely impaired phosphorylation of the adaptor protein linker for activation of T cells (LAT), a crucial component of the TCR-mediated signaling pathways. In healthy T lymphocytes, LAT is a membrane-bound protein and becomes phosphorylated by ζ-associated protein of 70 kDa (ZAP-70) upon TCR engagement. The molecular basis underlying the deficient phosphorylation of LAT and consequently the hyporesponsiveness of the synovial fluid T lymphocytes lies in the membrane displacement of LAT. We demonstrate that the subcellular localization of LAT is sensitive to changes in the intracellular levels of the antioxidant glutathione. The membrane anchorage of LAT, and consequently the phosphorylation of LAT and the cellular activation of the synovial fluid T lymphocytes upon TCR engagement, is restored in synovial fluid T lymphocytes after supplementation of the intracellular glutathione levels with N-acetyl-l-cysteine. These data suggest a role for the membrane displacement of LAT in the hyporesponsiveness of the synovial fluid T lymphocytes as a consequence of oxidative stress.

Objective To examine the expression of the thioredoxin (TRX)–thioredoxin reductase (TR) system in patients with rheumatoid arthritis (RA) and patients with other rheumatic diseases. Methods Levels of TRX in plasma and synovial fluid (SF) were measured using enzyme-linked immunosorbent assay. Cellular distribution of TRX was determined by flow cytometry and histochemistry. Cellular expression of TR was studied by in situ messenger RNA (mRNA) hybridization. The effect of oxidative stress and tumor necrosis factor α (TNFα) on TRX expression by cultured rheumatoid fibroblast-like synoviocytes was studied. Results Significantly increased TRX levels were found in the SF from 22 patients with RA, when compared with plasma levels in the same patients (P < 0.001) and compared with SF TRX levels in 15 patients with osteoarthritis (P < 0.001), 13 patients with gout (P < 0.05), and 9 patients with reactive arthritis (P < 0.0001). The presence of TRX could be demonstrated within the SF-derived mononuclear cells and synovial tissue (ST) of RA patients. Concordantly, expression of TR mRNA was observed in the ST of these patients. Stimulation of synovial fibroblast-like synoviocytes with either H2O2 or TNFα induced an increase in the production of TRX. Conclusion The data demonstrate significantly increased concentrations of TRX in the SF and ST of RA patients when compared with the levels in patients with other joint diseases. Evidence is presented that the local environment in the rheumatic joint contributes to increased TRX production. Based on its growth-promoting and cytokine-like properties, it is proposed that increased expression of TRX contributes to the disease activity in RA.

CD5 acts as a coreceptor on T lymphocytes and plays an important role in T-cell signaling and T-cell-B-cell interactions. Costimulation of T lymphocytes with anti-CD5 antibodies results in an increase of the intracellular Ca2+ levels, and subsequently in the activation of Ca2+/calmodulin-dependent (CaM) kinase type IV. In the present study, we have characterized the initial signaling pathway induced by anti-CD5 costimulation. The activation of phosphatidylinositol (PI) 3-kinase through tyrosine phosphorylation of its p85 subunit is a proximal event in the CD5-signaling pathway and leads to the activation of the lipid kinase activity of the p110 subunit. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit the CDS-induced response as assessed in interleukin-2 (IL-2) secretion experiments. The expression of an inactivated; Rac1 mutant (Rac1 . N17) in T lymphocytes transfected with an IL-2 promoter-driven reporter construct also abrogates the response to CD5 costimulation, while the expression of a constitutively active Rac1 mutant (Rac1-V12) completely replaces the CD5 costimulatory signal. The Rac1-specific guanine nucleotide exchange factor Vav is heavily phosphorylated on tyrosine residues upon CD5 costimulation, which is a prerequisite for its activation. A role for Vav in the CD5-induced signaling pathway is further supported by the findings that the expression of a dominant negative Vav mutant (Vav-C) completely abolishes the response to CD5 costimulation while the expression of a constitutively active Vav mutant [Vav(Delta 1-65)] makes the CD5 costimulation signal superfluous. Wortmannin is unable to block the Vav(Delta 1-65)- or Rac1 . V12-induced signals, indicating that both Vav and Rac1 function downstream from PI 3-kinase. Vav and Rac1 both act upstream from the CD5-induced activation df CaM kinase IV, since KN-62, an inhibitor of CaM kinases, and a dominant negative CaM kinase IV mutant block the Vav(Delta 1-65)- and Rac1 . V12-mediated signals. We propose a model for the CDS-induced signaling pathway in which the PI 3-kinase lipid products, together with tyrosine phosphorylation, activate Vav, resulting in the activation of Rac1 by the Vav-mediated exchange of GDP for GTP.

SummaryDendritic 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.

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, IKKe and CYLD are recruited to phosphorylated LSP1. IKKe 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 IKKe-CYLD-dependent signalling pathway leading to Bcl3 activation, which might be targeted in vaccination strategies or to prevent aberrant inflammation and allergy.

Innate immune cells such as dendritic cells (DCs), Langerhans cells (LCs), and macrophages are equipped with pattern recognition receptors that sense invading pathogens and activate immune responses. Recognition of the heavily glycosylated human immunodeficiency virus type-1 (HIV-1) envelope proteins by innate immune cells is mediated through membrane-bound C-type lectin receptors (CLRs) that interact with the carbohydrate structures. In addition, soluble lectin receptors present in tissue or blood can also bind to HIV-1 glycans. Capture of HIV-1 through CLRs is crucial for antigen presentation and induction of antiviral immunity. Strikingly, HIV-1 has evolved to exploit these innate receptors to facilitate infection as well as promote transmission to CD4+ T cells. The outcome of HIV-1-glycan recognition by the host is strongly dependent on the cell type and receptors involved. Identification of the molecular mechanisms and functional results of glycan-mediated recognition of HIV-1 is essential for a better understanding of HIV-1 pathogenesis and will lead to the development of novel antiviral strategies. Here, we discuss the current knowledge on recognition of HIV-1 through innate lectin receptors and the implications for viral replication, transmission, and immunity.

Host-pathogen interactions have coevolved for many years. On the one hand, the human immune system consists of innate and adaptive immune cells that function to defeat pathogens, and on the other hand, pathogens have coevolved to use the system for their own propagation. C-type lectins are conserved receptors recognizing carbohydrate structures on viruses, bacteria, parasites, and fungi. C-type lectins such as DC-SIGN, langerin, and dectin-1 are expressed by dendritic cell subsets and macrophages. Pathogen recognition by C-type lectins triggers signaling pathways that lead to the expression of specific cytokines which subsequently instruct adaptive T helper immune responses. T helper cell differentiation is crucial for initiating proper adaptive immune responses; some pathogens, however, use pattern recognition receptors like C-type lectins to subvert immune responses for survival. This review provides an update on the role of C-type lectins in HIV-1, mycobacterial, and Candida infections, and the coevolution of hosts and pathogens.

C-type lectins dectin-1 and dectin-2 on dendritic cells elicit protective immunity against fungal infections through induction of TH1 and TH-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 TH-17 immunity by selective activation of NF-κB subunit c-Rel, which induces expression of TH-17-polarizing cytokines IL-1β and IL-23p19. Malt1 inhibition abrogates c-Rel activation and TH-17 immunity to Candida species. We found that Malt1-mediated activation of c-Rel is similarly essential to induction of TH-17-polarizing cytokines by dectin-2. Whereas dectin-1 activates all NF-κB subunits, dectin-2 selectively activates c-Rel, signifying a specialized TH-17-enhancing function for dectin-2 in anti-fungal immunity by human dendritic cells. Thus, dectin-1 and dectin-2 control adaptive TH-17 immunity to fungi via Malt1-dependent activation of c-Rel., Author Summary Fungal infections are a major health threat and the incidence is growing worldwide. There is a need for efficient antifungal vaccines. Adaptive immune responses and in particular T helper cell type 17 (TH-17) responses are crucial in the defence against fungal infections. Human dendritic cells (DCs) induce TH-17 responses after interaction with fungi. DCs express C-type lectins dectin-1 and dectin-2 that interact with the carbohydrate structures present in the cell-wall of fungi. It is unclear how signaling by these C-type lectins leads to specific TH-17 responses. Here we demonstrate that the signaling molecule Malt1 present in the CARD9-Bcl10-Malt1 complex is responsible for TH-17 induction by selectively activating the NF-κB transcription factor c-Rel, which drives transcription of the TH-17-polarizing cytokines. Inhibition of either Malt1 or c-Rel prevents TH-17 induction in response to fungi. Furthermore, we show that the C-type lectin dectin-2 selectively activates c-Rel, signifying a specialized TH-17-enhancing function for this C-type lectin. Thus, novel vaccination strategies that target dectin-2 or activate Malt1 can induce predominant TH-17 responses. Since aberrant TH-17 responses underlie the pathology of atopic dermatitis and various autoimmune diseases, Malt1 is a rational therapeutic target to attenuate anomalous adaptive immune responses.

Purpose of review This review summarizes the current knowledge of innate signaling events that are involved in HIV-1 infection. We here focus on dendritic cells, which are among the first cells that encounter HIV-1 after exposure. Recent findings HIV-1 triggers multiple pattern recognition receptors on dendritic cells that facilitate infection and transmission to T cells. Triggering of the C-type lectin DC-SIGN induces signals that promote HIV-1 replication in dendritic cells and transmission to T cells. Similarly, dendritic cell immunoreceptor has been shown to bind HIV-1 and facilitate transmission to T cells. The cytosolic sensors TRIM5 and cyclophilin A recognize capsid proteins and activate antiviral responses to prevent HIV-1 infection. Moreover, activation of mammalian target of rapamycin (mTOR) by HIV downregulates autophagy preventing adaptive immune responses. Summary Dendritic cells express an array of pattern recognition receptors that are involved in HIV-1 infection. However, HIV-1 dampens signaling by these receptors leading to suppressed responses or takes advantage of their signaling for its own benefit.

Pathogen recognition is central to the induction of adaptive immunity. Dendritic cells (DCs) express different pattern recognition receptors (PRRs), such as Toll-like receptors and C-type lectins, that sense invading pathogens. Pathogens trigger a specific set of PRRs, leading to activation of intracellular signaling processes that shapes the adaptive immunity. It is becoming clear that cross talk between these signaling routes is crucial for pathogen-tailored immune responses. The C-type lectin DC-SIGN interacts with different mannose-expressing pathogens such as Mycobacterium tuberculosis and HIV-1. Notably, DC-SIGN triggering by these pathogens results in a specific Raf-1-dependent signaling pathway that modulates TLR-induced NF-kappaB activation. Here, we will discuss the various methods that we have used to identify the innate signaling by the C-type lectin DC-SIGN, and how to analyze the consequences on NF-kappaB activation.

Pathogen recognition by dendritic cells (DCs) is central to the induction of adaptive immunity. Pattern recognition receptors (PRRs) on DCs interact with pathogens, leading to signaling events that dictate adaptive immune responses. It is becoming clear that C-type lectins are important PRRs that recognize carbohydrate structures. Most pathogens express carbohydrate structures on their surface and these function as a so-called sugar fingerprint that are recognized by specific C-type lectins. Triggering of C-type lectin induces signaling cascades that initiate or modulate specific cytokine responses and therefore tailor T cell polarization to the pathogens. Here we will discuss our current understanding of the innate signaling pathways induced by C-type lectins DC-SIGN and dectin-1 in humans and how these pathways shape adaptive immunity.

Pathogen recognition is central to the induction of adaptive immunity. Dendritic cells (DCs) express different pattern recognition receptors (PRRs), such as Toll-like receptors and C-type lectins, that sense invading pathogens. Pathogens trigger a specific set of PRRs, leading to activation of intracellular signaling processes that shapes the adaptive immunity. It is becoming clear that cross talk between these signaling routes is crucial for pathogen-tailored immune responses. The C-type lectin DC-SIGN interacts with different mannose-expressing pathogens such as Mycobacterium tuberculosis and HIV-1. Notably, DC-SIGN triggering by these pathogens results in a specific Raf-1-dependent signaling pathway that modulates TLR-induced NF-kappaB activation. Here, we will discuss the various methods that we have used to identify the innate signaling by the C-type lectin DC-SIGN, and how to analyze the consequences on NF-kappaB activation.

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-kappa B 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-kappa B 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

Dendritic cells (DCs) tailor adaptive immune responses to specific pathogens. This diversity is mediated by cooperation between different pattern recognition receptors that are triggered by specific pathogens. DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is a pattern recognition receptor with a broad pathogen recognition specificity as a result of its affinity for mannose and fucose carbohydrates. DC-SIGN induces very diverse immune responses to different pathogens, such as bacteria, fungi, helminths and viruses. Recent data show that DC-SIGN triggering by pathogens modulates Toll-like receptor signaling at the level of nuclear factor-κB. In this article, we will discuss the signaling pathways induced by DC-SIGN and its central role in the regulation of adaptive immunity to bacterial, fungal and viral pathogens.

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-kappa B 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

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.

Adaptive immune responses by dendritic cells (DCs) are critically controlled by Toll-like receptor (TLR) function. Little is known about modulation of TLR-specific signaling by other pathogen receptors. Here, we have identified a molecular signaling pathway induced by the C-type lectin DC-SIGN that modulates TLR signaling at the level of the transcription factor NF-kappaB. We demonstrated that pathogens trigger DC-SIGN on human DCs to activate the serine and threonine kinase Raf-1, which subsequently leads to acetylation of the NF-kappaB subunit p65, but only after TLR-induced activation of NF-kappaB. Acetylation of p65 both prolonged and increased IL10 transcription to enhance anti-inflammatory cytokine responses. We demonstrated that different pathogens such as Mycobacterium tuberculosis, M. leprae, Candida albicans, measles virus, and human immunodeficiency virus-1 interacted with DC-SIGN to activate the Raf-1-acetylation-dependent signaling pathway to modulate signaling by different TLRs. Thus, this pathway is involved in regulation of adaptive immunity by DCs to bacterial, fungal, and viral pathogens.

Dendritic cells are crucial in pathogen recognition and induction of specific immune responses to eliminate pathogens from the infected host. Host recognition of invading microorganisms relies on evolutionarily conserved, germline-encoded pattern-recognition receptors (PRRs) that are expressed by DCs. The best-characterized PRR family comprises the Toll-like receptors (TLRs) that recognize bacteria or viruses. In addition to TLRs, intracellular Nod-like receptors and the membrane-associated C-type lectins (CLRs) function as PRRs. Many of these innate receptors also have an important function in natural host homeostatic responses, such as the maintenance of gut homeostasis. Clearly, more indications are hinting at a fine-tuning of immune responses by a concerted action of these PRRs on the recognition of pathogen components and the consequent signalling events that are created. It is becoming increasingly clear that these PRRs can initiate specific signalling events that modulate the production of inflammatory cytokines, phagocytosis, intracellular routing of antigen, release of oxidative species and DC maturation and the subsequent development of adaptive immunity. Notably, members within one family of PRRs can trigger opposite signalling features, indicating that the ultimate outcome of pathogen-induced immune responses depends on the pathogen signature and the collective PRRs involved.

Homeostatic control of T cells involves tight regulation of effector T cells to prevent excessive activation that can cause tissue damage and autoimmunity. Little is known, however, about whether antigen-presenting cells (APCs) are also involved in maintaining immune system homeostasis once effector T cells are stimulated. Here we found that immature APCs downregulated effector T cell function by a mechanism involving the C-type lectin MGL expressed by APCs. Glycosylation-dependent interactions of MGL with CD45 on effector T cells negatively regulated T cell receptor-mediated signaling and T cell-dependent cytokine responses, which in turn decreased T cell proliferation and increased T cell death. Thus, regulation of effector T cells by MGL expressed on APCs may provide a target for regulating chronic inflammatory and autoimmune diseases.

Glycan molecules covalently linked to proteins or lipids control vital properties of cells, such as signaling, adherence, and migration through the body. The biosynthesis of such glycans depends on the concerted action of many endoplasmic reticulum and Golgi enzymes, a process that is tightly ordered and regulated. To understand the function of glycoconjugates in cellular interactions, it is crucial to investigate the regulation of expression of the genes encoding the "glycosylation-related" genes, encompassing large families of glycosyltransferases, glycosidases, and sulfotransferases. This chapter describes an easy, flexible, and reliable method of quantitative real-time polymerase chain reaction to measure the expression levels of 80 human glycosylation-related genes that primarily encode common enzymes involved in N- and O-linked protein glycosylation and/or glycolipids. Designing and including additional primer sets to detect more genes can easily extend the system. In order to allow the normalization of gene expression data obtained by real-time polymerase chain reaction within different cells, tissues, or under different experimental conditions, a protocol is included to detect genes suitable for use as endogenous reference genes.