Characterization of polarization states of canine monocyte derived macrophages.

Macrophages can reversibly polarize into multiple functional subsets depending on their micro-environment. Identification and understanding the functionality of these subsets is relevant for the study of immune­related diseases. However, knowledge about canine macrophage polarization is still in its infancy. In this study, we polarized canine monocytes using GM-CSF/IFN- γ and LPS towards M1 macrophages or M-CSF and IL-4 towards M2 macrophages and compared them to undifferentiated monocytes (M0). Polarized M1 and M2 macrophages were thoroughly characterized for morphology, surface marker features, gene profiles and functional properties. Our results showed that canine M1-polarized macrophages obtained a characteristic large, roundish, or amoeboid shape, while M2-polarized macrophages were smaller and adopted an elongated spindle-like morphology. Phenotypically, all macrophage subsets expressed the pan-macrophage markers CD14 and CD11b. M1-polarized macrophages expressed increased levels of CD40, CD80 CD86 and MHC II, while a significant increase in the expression levels of CD206, CD209, and CD163 was observed in M2-polarized macrophages. RNAseq of the three macrophage subsets showed distinct gene expression profiles, which are closely associated with immune responsiveness, cell differentiation and phagocytosis. However, the complexity of the gene expression patterns makes it difficult to assign clear new polarization markers. Functionally, undifferentiated -monocytes, and M1- and M2- like subsets of canine macrophages can all phagocytose latex beads. M2-polarized macrophages exhibited the strongest phagocytic capacity compared to undifferentiated monocytes- and M1-polarized cells. Taken together, this study showed that canine M1 and M2-like macrophages have distinct features largely in parallel to those of well-studied species, such as human, mouse and pig. These findings enable future use of monocyte derived polarized macrophages particularly in studies of immune related diseases in dogs.

Immune Responses and Pathogenesis following Experimental SARS-CoV-2 Infection in Domestic Cats.

Several reports demonstrated the susceptibility of domestic cats to SARS-CoV-2 infection. Here, we describe a thorough investigation of the immune responses in cats after experimental SARS-CoV-2 inoculation, along with the characterization of infection kinetics and pathological lesions. Specific pathogen-free domestic cats (n = 12) were intranasally inoculated with SARS-CoV-2 and subsequently sacrificed on DPI (days post-inoculation) 2, 4, 7 and 14. None of the infected cats developed clinical signs. Only mild histopathologic lung changes associated with virus antigen expression were observed mainly on DPI 4 and 7. Viral RNA was present until DPI 7, predominantly in nasal and throat swabs. The infectious virus could be isolated from the nose, trachea and lungs until DPI 7. In the swab samples, no biologically relevant SARS-CoV-2 mutations were observed over time. From DPI 7 onwards, all cats developed a humoral immune response. The cellular immune responses were limited to DPI 7. Cats showed an increase in CD8+ cells, and the subsequent RNA sequence analysis of CD4+ and CD8+ subsets revealed a prominent upregulation of antiviral and inflammatory genes on DPI 2. In conclusion, infected domestic cats developed a strong antiviral response and cleared the virus within the first week after infection without overt clinical signs and relevant virus mutations.

Leucinostatin acts as a co-inducer for heat shock protein 70 in cultured canine retinal pigment epithelial cells.

Dysregulation of retinal pigment epithelium (RPE) cells is the main cause of a variety of ocular diseases. Potentially heat shock proteins, by preventing molecular and cellular damage and modulating inflammatory disease, may exert a protective role in eye disease. In particular, the inducible form of heat shock protein 70 (Hsp70) is widely upregulated in inflamed tissues, and in vivo upregulation of Hsp70 expression by HSP co-inducing compounds has been shown to be a potential therapeutic strategy for inflammatory diseases. In order to gain further understanding of the potential protective effects of Hsp70 in RPE cells, we developed a method for isolation and culture of canine RPE cells. Identity of RPE cells was confirmed by detection of its specific marker, RPE65, in qPCR, flow cytometry, and immunocytochemistry analysis. The ability of RPE cells to express Hsp70 upon experimental induction of cell stress, by arsenite, was analyzed by flow cytometry. Finally, in search of a potential Hsp70 co-inducer, we investigated whether the compound leucinostatin could enhance Hsp70 expression in stressed RPE cells. Canine RPE cells were isolated and cultured successfully. Purity of cells that strongly expressed RPE65 was over 90%. Arsenite-induced stress led to a time- and dose-dependent increase in Hsp70 expression in canine RPE cells in vitro. In addition, leucinostatin, which enhanced heat shock factor-1-induced transcription from the heat shock promoter in DNAJB1-luc-O23 reporter cell line, also enhanced Hsp70 expression in arsenite-stressed RPE cells, in a dose-dependent fashion. These findings demonstrate that leucinostatin can boost Hsp70 expression in canine RPE cells, most likely by activating heat shock factor-1, suggesting that leucinostatin might be applied as a new co-inducer for Hsp70 expression.

Matured Tolerogenic Dendritic Cells Effectively Inhibit Autoantigen Specific CD4+ T Cells in a Murine Arthritis Model.

Tolerogenic dendritic cells (tolDCs) are a promising treatment modality for diseases caused by a breach in immune tolerance, such as rheumatoid arthritis. Current medication for these diseases is directed toward symptom suppression but no real cure is available yet. TolDC-based therapy aims to restore immune tolerance in an antigen-specific manner. Here we used a mouse model to address two major questions: (i) is a maturation stimulus needed for tolDC function in vitro and in vivo and is maturation required for functioning in experimental arthritis and (ii) can tolDCs modulate CD4+ T cell responses? To answer these questions, we compared matured and immature dexamethasone/vitamin D3-generated tolDCs in vitro. Subsequently, we co-transferred these tolDCs with naïve or effector CD4+ T cells to study the characteristics of transferred T cells after 3 days with flow cytometry and Luminex multiplex assays. In addition, we tested the suppressive capabilities of tolDCs in an experimental arthritis model. We found that tolDCs cannot only modulate naïve CD4+ T cell responses as shown by fewer proliferated and activated CD4+ T cells in vivo, but also effector CD4+ T cells. In addition, Treg (CD4+CD25+FoxP3+) expansions were seen in the proliferating cell population in the presence of tolDCs. Furthermore, we show that administered tolDCs are capable to inhibit arthritis in the proteoglycan-induced arthritis model. However, a maturation stimulus is needed for tolDCs to manifest this tolerizing function in an inflammatory environment. Our data will be instrumental for optimization of future tolDC therapies for autoimmune diseases.

Heat Shock Proteins Can Be Surrogate Autoantigens for Induction of Antigen Specific Therapeutic Tolerance in Rheumatoid Arthritis.

Technologies that enable induction of therapeutic tolerance may revolutionize the treatment of autoimmune diseases by their supposed potential to induce drug-free and lasting disease remission. In combination with diagnostic tests that screen for individuals at risk, these approaches may offer chances to halt disease before serious damage in the tissues can occur. In fact, for healthy individuals at risk, this could lead to a preventive form of vaccination. For therapeutic tolerance to re-instate natural self-tolerance it seems essential to induce tolerance for the critical autoantigens involved in disease. However, for most autoimmune diseases such antigens are poorly defined. This is the case for both disease inciting autoantigens and antigens that become involved through epitope spreading. A possible source of surrogate auto-antigens expressed in tissues during inflammation are heat shock proteins (HSP) or stress proteins. In this mini-review we discuss unique characteristics of HSP which provide them with the capacity to inhibit inflammatory processes. Various studies have shown that epitopes of HSP60 and HSP70 molecules can function as vaccines to downregulate a variety of autoimmune inflammatory diseases. Currently, several research groups are developing cell therapies with the intention to reach therapeutic tolerance. In this review, in which we are proposing to ex vivo load tolerant dendritic cells with a Treg inducing HSP70 derived peptide called B29, we are discussing the chances to develop this as an autologous tolDC therapeutic tolerance therapy for rheumatoid arthritis.

Lactobacillus rhamnosus GG-Derived Soluble Mediators Modulate Adaptive Immune Cells.

Probiotics and probiotic-related nutritional interventions have been described to have beneficial effects on immune homeostasis and gut health. In previous studies, Lactobacillus rhamnosus GG (LGG) soluble mediators (LSM) have been demonstrated to exert beneficial effects in preclinical models of allergic sensitization, bacterial infection, and intestinal barrier function. In the context of allergic diseases, differentiation of dendritic cells (DCs) and their interactions with T cell populations are crucial for driving tolerogenic responses. In this study, we set out to evaluate whether these LSM can modulate DC maturation and have an impact on prompting protective and/or tolerogenic T cell responses. Monocytes were isolated from PBMC of healthy blood donors and cultured in the presence of GM-CSF, IL-4, and LSM or unconditioned bacterial culture medium control (UCM) during 6 days to induce DC differentiation. Subsequently, these DCs were matured in the presence of TNF-α for 1 day and analyzed for their phenotype and ability to induce autologous T cell activation and differentiation to model recall antigens. After 7 days of co-culture, T cells were analyzed for activation and differentiation by flow cytometry of intracellular cytokines (IFN-γ, IL-2, IL-10, and IL-17A), activation markers (CD25), and Foxp3+ expression. LSM did not alter DC numbers or maturation status. However, these DCs did show improved capacity to induce a T cell response as shown by increased IL-2 and IFN-γ producing T cell populations upon stimulation with recall antigens. These enhanced recall responses coincided with enhanced Foxp3+ expression that was not observed when T cells were cultured in the presence of UCM-treated DCs. By contrast, the number of activated T cells (determined by CD25 expression) was only slightly increased. In conclusion, this study reveals that LSM can influence adaptive immune responses as shown by the modulation of DC functionality. These mechanisms might contribute to previous observed effects in animal models in vivo. Altogether, these results suggest that LSM may provide an alternative to live probiotics in case life bacteria may not be used because of health conditions, although further clinical testing is needed.

Routing dependent immune responses after experimental R848-adjuvated vaccination.

Most traditional vaccines are administered via the intramuscular route. Other routes of administration however, can induce equal or improved protective memory responses and might provide practical advantages such as needle-free immunization, dose sparing and induction of tissue-specific (mucosal) immunity. Here we explored the differences in immunological outcome after immunization with model antigens via two promising immunization routes (intradermal and intranasal) with or without the experimental adjuvant and TLR7/8-agonist R848. Because the adaptive immune response is largely determined by the local innate cells at the site of immunization, the effect of R848-adjuvation on local cellular recruitment, antigenic uptake by antigen-presenting cells and the initiation of the adaptive response were analyzed for the two routes of administration. We show a general immune-stimulating effect of R848 irrespective of the route of administration. This includes influx of neutrophils, macrophages and dendritic cells to the respective draining lymph nodes and an increase in antigen-positive antigen-presenting cells which leads for both intradermal and intranasal immunization to a mainly TH1 response. Furthermore, both intranasal and intradermal R848-adjuvated immunization induces a local shift in DC subsets; frequencies of CD11b+DC increase whereas CD103+DC decrease in relative abundance in the draining lymph node. In spite of these similarities, the outcome of immune responses differs for the respective immunization routes in both magnitude and cytokine profile. Via the intradermal route, the induced T-cell response is higher compared to that after intranasal immunization, which corresponds with the local higher uptake of antigen by antigen-presenting cells after intradermal immunization. Furthermore, R848-adjuvation enhances ex vivo IL-10 and IL-17 production after intranasal, but not intradermal, T-cell activation. Quite the opposite, intradermal immunization leads to a decrease in IL-10 production by the vaccine induced T-cells. This knowledge may lead to a more rational development of novel adjuvanted vaccines administered via non-traditional routes.

The Immunomodulatory Potential of tolDCs Loaded with Heat Shock Proteins.

Disease suppressive T cell regulation may depend on cognate interactions of regulatory T cells with self-antigens that are abundantly expressed in the inflamed tissues. Heat shock proteins (HSPs) are by their nature upregulated in stressed cells and therefore abundantly present as potential targets for such regulation. HSP immunizations have led to inhibition of experimentally induced inflammatory conditions in various models. However, re-establishment of tolerance in the presence of an ongoing inflammatory process has remained challenging. Since tolerogenic DCs (tolDCs) have the combined capacity of mitigating antigen-specific inflammatory responses and of endowing T cells with regulatory potential, it seems attractive to combine the anti-inflammatory qualities of tolDCs with those of HSPs.

Regulatory T cell frequencies and phenotypes following anti-viral vaccination.

Regulatory T cells (Treg) function in the prevention of excessive inflammation and maintenance of immunological homeostasis. However, these cells may also interfere with resolution of infections or with immune reactions following vaccination. Effects of Treg on vaccine responses are nowadays investigated, but the impact of vaccination on Treg homeostasis is still largely unknown. This may be a relevant safety aspect, since loss of tolerance through reduced Treg may trigger autoimmunity. In exploratory clinical trials, healthy adults were vaccinated with an influenza subunit vaccine plus or minus the adjuvant MF59®, an adjuvanted hepatitis B subunit vaccine or a live attenuated yellow fever vaccine. Frequencies and phenotypes of resting (rTreg) and activated (aTreg) subpopulations of circulating CD4+ Treg were determined and compared to placebo immunization. Vaccination with influenza vaccines did not result in significant changes in Treg frequencies and phenotypes. Vaccination with the hepatitis B vaccine led to slightly increased frequencies of both rTreg and aTreg subpopulations and a decrease in expression of functionality marker CD39 on aTreg. The live attenuated vaccine resulted in a decrease in rTreg frequency, and an increase in expression of activation marker CD25 on both subpopulations, possibly indicating a conversion from resting to migratory aTreg due to vaccine virus replication. To study the more local effects of vaccination on Treg in lymphoid organs, we immunized mice and analyzed the CD4+ Treg frequency and phenotype in draining lymph nodes and spleen. Vaccination resulted in a transient local decrease in Treg frequency in lymph nodes, followed by a systemic Treg increase in the spleen. Taken together, we showed that vaccination with vaccines with an already established safe profile have only minimal impact on frequencies and characteristics of Treg over time. These findings may serve as a bench-mark of inter-individual variation of Treg frequencies and phenotypes following vaccination.

Bystander activation of irrelevant CD4+ T cells following antigen-specific vaccination occurs in the presence and absence of adjuvant.

Autoimmune and other chronic inflammatory diseases (AID) are prevalent diseases which can severely impact the quality of life of those that suffer from the disease. In most cases, the etiology of these conditions have remained unclear. Immune responses that take place e.g. during natural infection or after vaccination are often linked with the development or exacerbation of AID. It is highly debated if vaccines induce or aggravate AID and in particular adjuvants are mentioned as potential cause. Since vaccines are given on a large scale to healthy individuals but also to elderly and immunocompromised individuals, more research is warranted. Non-specific induction of naïve or memory autoreactive T cells via bystander activation is one of the proposed mechanisms of how vaccination might be involved in AID. During bystander activation, T cells unrelated to the antigen presented can be activated without (strong) T cell receptor (TCR) ligation, but via signals derived from the ongoing response directed against the vaccine-antigen or adjuvant at hand. In this study we have set up a TCR transgenic T cell transfer mouse model by which we were able to measure local bystander activation of transferred and labeled CD4+ T cells. Intramuscular injection with the highly immunogenic Complete Freund's Adjuvant (CFA) led to local in vivo proliferation and activation of intravenously transferred CD4+ T cells in the iliac lymph node. This local bystander activation was also observed after CFA prime and Incomplete Freund's Adjuvant (IFA) boost injection. Furthermore, we showed that an antigen specific response is sufficient for the induction of a bystander activation response and the general, immune stimulating effect of CFA or IFA does not appear to increase this effect. In other words, no evidence was obtained that adjuvation of antigen specific responses is essential for bystander activation.

Oral exposure to drugs with immune-adjuvant potential induces hypersensitivity responses to the reporter antigen TNP-OVA.

Immune-mediated drug hypersensitivity reactions are important causes of black box warnings and drug withdrawals. Despite the high demand for preclinical screening tools, no validated in vitro or in vivo models are available. In the current study, we used a previously described oral administration model using trinitrophenyl-ovalbumin (TNP-OVA) as an antigen to report immuno-adjuvating effects of the analgesic drug acetaminophen (APAP) and its nonhepatotoxic regioisomer 3'-hydroxyacetanilide (AMAP), the antibiotic ofloxacin (OFLX), the antiepileptic drug carbamazepine (CMZ), and the antidiabetic drug metformin (MET). Furthermore, APAP and AMAP were tested in a popliteal lymph node assay (PLNA) combined with TNP-OVA as reporter antigen (RA). C3H/HeOuJ mice were dosed by oral gavage with diclofenac (DF), APAP, AMAP, OFLX, MET, or CMZ. On the first exposure day, the mice received an ip injection with TNP-OVA. Fifteen days later, they were ear challenged with TNP-OVA and delayed-type hypersensitivity (DTH) responses were assessed 24 h later. One week after challenge, the ear-draining lymph node was removed and TNP-specific antibody-secreting cells were determined. DF, APAP, CMZ, and OFLX showed a significant increase in DTH responses to ear injection with TNP-OVA, whereas AMAP and MET did not. C57BL/6 mice were slightly less responsive to APAP and DF after oral gavage, and importantly both AMAP and APAP were negative in the RA-PLNA. The present work shows that the oral exposure model using RA and the RA-PLNA may serve to screen the immune-adjuvant potential of new chemical entities during preclinical drug development.

An in vivo tiered approach to test immunosensitization by low molecular weight compounds.

New chemical entities are tested in general toxicity assays during development before entering clinical trials. However, immunosensitization of these entities is not tested on a standard basis. There are no in vitro or in vivo standardized methods available for testing immunosensitization or immunostimulation. In this chapter, we describe a tiered strategy oral exposure model for assessing immunosensitization or immunostimulation capacity of low molecular weight compounds. The strategy starts from a set of data that may provide information on bioactivation, conjugation (hapten-protein conjugate formation), cytotoxicity and signs of inflammation in any of the animals in a 28 day-toxicity study. In case of concern, a reporter antigen-popliteal lymph node assay (RA-PLNA) and, subsequently, an oral exposure experiment with the reporter antigen can be performed. Based on the presence of RA-specific immune responses an indication for immunosensitization can be found.

Bile salt-stimulated lipase from human milk binds DC-SIGN and inhibits human immunodeficiency virus type 1 transfer to CD4+ T cells.

A wide range of pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, Mycobacterium, Leishmania, and Helicobacter pylori, can interact with dendritic cell (DC)-specific ICAM3-grabbing nonintegrin (DC-SIGN), expressed on DCs and a subset of B cells. More specifically, the interaction of the gp120 envelope protein of HIV-1 with DC-SIGN can facilitate the transfer of virus to CD4+ T lymphocytes in trans and enhance infection. We have previously demonstrated that a multimeric LeX component in human milk binds to DC-SIGN, preventing HIV-1 from interacting with this receptor. Biochemical analysis reveals that the compound is heat resistant, trypsin sensitive, and larger than 100 kDa, indicating a specific glycoprotein as the inhibitory compound. By testing human milk from three different mothers, we found the levels of DC-SIGN binding and viral inhibition to vary between samples. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and matrix-assisted laser desorption ionization analysis, we identified bile salt-stimulated lipase (BSSL), a Lewis X (LeX)-containing glycoprotein found in human milk, to be the major variant protein between the samples. BSSL isolated from human milk bound to DC-SIGN and inhibited the transfer of HIV-1 to CD4+ T lymphocytes. Two BSSL isoforms isolated from the same human milk sample showed differences in DC-SIGN binding, illustrating that alterations in the BSSL forms explain the differences observed. These results indicate that variations in BSSL lead to alterations in LeX expression by the protein, which subsequently alters the DC-SIGN binding capacity and the inhibitory effect on HIV-1 transfer. Identifying the specific molecular interaction between the different forms may aid in the future design of antimicrobial agents.

Two way communication between neutrophils and dendritic cells.

Neutrophils are instrumental in innate immunity by mediating immediate removal of pathogens. Recent findings demonstrate that they are able to communicate with dendritic cells, which form a link between innate and adaptive immunity by stimulating long-term memory T cell responses. This communication occurs through the cell-surface receptors Mac-1 and carcinoembryonic antigen-related cellular adhesion molecule on neutrophils, which interact with C-type lectins such as dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN). Clearly, dendritic cells can prolong the lifespan of neutrophils, whereas neutrophils could direct dendritic cell-mediated T cell responses.

Lewis X component in human milk binds DC-SIGN and inhibits HIV-1 transfer to CD4+ T lymphocytes.

DC-specific ICAM3-grabbing non-integrin (DC-SIGN), which is expressed on DCs, can interact with a variety of pathogens such as HIV-1, hepatitis C, Ebola, cytomegalovirus, Dengue virus, Mycobacterium, Leishmania, and Candida albicans. We demonstrate that human milk can inhibit the DC-SIGN-mediated transfer of HIV-1 to CD4+ T lymphocytes as well as viral transfer by both immature and mature DCs. The inhibitory factor directly interacted with DC-SIGN and prevented the HIV-1 gp120 envelope protein from binding to the receptor. The human milk proteins lactoferrin, alpha-lactalbumin, lysozyme, beta-casein, and secretory leukocyte protease inhibitor did not bind DC-SIGN or demonstrate inhibition of viral transfer. The inhibitory effect could be fully alleviated with an Ab recognizing the Lewis X (LeX) sugar epitope, commonly found in human milk. LeX in polymeric form or conjugated to protein could mimic the inhibitory activity, whereas free LeX sugar epitopes could not. We reveal that a LeX motif present in human milk can bind to DC-SIGN and thereby prevent the capture and subsequent transfer of HIV-1 to CD4+ T lymphocytes. The presence of such a DC-SIGN-binding molecule in human milk may both influence antigenic presentation and interfere with pathogen transfer in breastfed infants.

Interactions of DC-SIGN with Mac-1 and CEACAM1 regulate contact between dendritic cells and neutrophils.

Early during infection neutrophils are the most important immune cells that are involved in killing of pathogenic bacteria and regulation of innate immune responses at the site of infection. It has become clear that neutrophils also modulate adaptive immunity through interactions with dendritic cells (DCs) that are pivotal in the induction of T cell responses. Upon activation, neutrophils release TNF-alpha and induce maturation of DCs that enables these antigen-presenting cells to stimulate T cell proliferation and to induce T helper 1 polarization. DC maturation by neutrophils also requires cellular interactions that are mediated by binding of the DC-specific receptor DC-SIGN to Mac-1 on the neutrophil. Here, we demonstrate that also CEACAM1 is an important ligand for DC-SIGN on neutrophils. Binding of DC-SIGN to both CEACAM1 and Mac-1 is required to establish cellular interactions with neutrophils. DC-SIGN is a C-type lectin that has specificity for Lewis(x), and we show that DC-SIGN mediates binding to CEACAM1 through Lewis(x) moieties that are specifically expressed on CEACAM1 derived from neutrophils. This indicates that glycosylation-driven binding of both Mac-1 and CEACAM1 to DC-SIGN is essential for interactions of neutrophils with DCs and enables neutrophils to modulate T cell responses through interactions with DCs.

Carbohydrate specificities of the murine DC-SIGN homologue mSIGNR1.

C-type lectins are important receptors expressed by antigen presenting cells that are involved in cellular communications as well as in pathogen uptake. An important C-type lectin family is represented by DC-SIGN and its homologues in human and mouse. Here we have investigated the carbohydrate specificity of cellular mSIGNR1 and compared it with DC-SIGN and L-SIGN. mSIGNR1 has a similar specificity as human DC-SIGN for high mannose-containing ligands present on both cellular and pathogen ligands. However, the DC-SIGN molecules differ in their recognition of Lewis antigens; mSIGNR1 interacts not only with Le(x/y) and Le(a/b) antigens similar to DC-SIGN, but also with sialylated Lex, a ligand for selectins. The differential recognition of Lewis antigens suggests differences between mSIGNR1 and DC-SIGN in the recognition of cellular ligands and pathogens that express Lewis epitopes.

Potency of HIV-1 envelope glycoprotein gp120 antibodies to inhibit the interaction of DC-SIGN with HIV-1 gp120.

The interaction of DC-SIGN with gp120 provides an attractive target for intervention of HIV-1 transmission. Here, we have investigated the potency of gp120 antibodies to inhibit the DC-SIGN-gp120 interaction. We demonstrate that although the V3 loop is not essential for DC-SIGN binding, antibodies against the V3 loop partially inhibit DC-SIGN binding, suggesting that these antibodies sterically hinder DC-SIGN binding to gp120. Polyclonal antibodies raised against non-glycosylated gp120 inhibited both low and high avidity DC-SIGN-gp120 interactions in contrast to polyclonal antibodies raised against glycosylated gp120. Thus, glycans present on gp120 may prevent the generation of antibodies that block the DC-SIGN-gp120 interactions. Moreover, the polyclonal antibodies against non-glycosylated gp120 efficiently inhibited HIV-1 capture by both DC-SIGN transfectants and immature dendritic cells. Therefore, non-glycosylated gp120 may be an attractive immunogen to elicit gp120 antibodies that block the binding to DC-SIGN. Furthermore, we demonstrate that DC-SIGN binding to gp120 enhanced CD4 binding, suggesting that DC-SIGN induces conformational changes in gp120, which may provide new targets for neutralizing antibodies.

Identification of the mycobacterial carbohydrate structure that binds the C-type lectins DC-SIGN, L-SIGN and SIGNR1.

Mycobacterium tuberculosis represents a worldwide health risk and although macrophages are primarily infected, dendritic cells (DC) are important in inducing cellular immune responses against M. tuberculosis. Recent studies have demonstrated that M. tuberculosis targets the DC-specific C-type lectin DC-SIGN to inhibit the immuno-stimulatory function of DC through the interaction of the mycobacterial mannosylated lipoarabinomannan (ManLAM) to DC-SIGN, which prevents DC maturation and induces the immuno-suppressive cytokine IL-10. This may contribute to survival and persistence of M. tuberculosis. Here, we have identified the specific pathogen-derived carbohydrate structure on ManLAM that is recognized by DC-SIGN. We have synthesized the mannose-cap oligosaccharides man-ara, (man)2-ara and (man)3-ara, and demonstrate that these neoglycoconjugates are specifically bound by DC-SIGN. Moreover, we demonstrate that the human and murine DC-SIGN homologue L-SIGN and SIGNR1, respectively, also interact with mycobacteria through ManLAM. Both homologues have the highest affinity for the (man)3-ara structure, similar to DC-SIGN. This study provides information about the specific carbohydrate structures on pathogens that are recognized by DC-SIGN, and may provide strategies to develop vaccines against these pathogens. Moreover, the identification of SIGNR1 as a receptor for ManLAM will enable in vivo studies to investigate the role of DC-SIGN in M. tuberculosis pathogenesis.

Hepatitis C virus targets DC-SIGN and L-SIGN to escape lysosomal degradation.

Hepatitis C virus (HCV) is a major health problem. However, the mechanism of hepatocyte infection is largely unknown. We demonstrate that the dendritic cell (DC)-specific C-type lectin DC-SIGN and its liver-expressed homologue L-SIGN/DC-SIGNR are important receptors for HCV envelope glycoproteins E1 and E2. Mutagenesis analyses demonstrated that both HCV E1 and E2 bind the same binding site on DC-SIGN as the pathogens human immunodeficiency virus type 1 (HIV-1) and mycobacteria, which is distinct from the cellular ligand ICAM-3. HCV virus-like particles are efficiently captured and internalized by DCs through binding of DC-SIGN. Antibodies against DC-SIGN specifically block HCV capture by both immature and mature DCs, demonstrating that DC-SIGN is the major receptor on DCs. Interestingly, internalized HCV virus-like particles were targeted to nonlysosomal compartments within immature DCs, where they are protected from lysosomal degradation in a manner similar to that demonstrated for HIV-1. Lewis X antigen, another ligand of DC-SIGN, was internalized to lysosomes, demonstrating that the internalization pathway of DC-SIGN-captured ligands may depend on the structure of the ligand. Our results suggest that HCV may target DC-SIGN to "hide" within DCs and facilitate viral dissemination. L-SIGN, expressed by THP-1 cells, internalized HCV particles into similar nonlysosomal compartments, suggesting that L-SIGN on liver sinusoidal endothelial cells may capture HCV from blood and transmit it to hepatocytes, the primary target for HCV. We therefore conclude that both DCs and liver sinusoidal endothelial cells may act as reservoirs for HCV and that the C-type lectins DC-SIGN and L-SIGN, as important HCV receptors, may represent a molecular target for clinical intervention in HCV infection.