key: cord-322617-znvikfza authors: Caparrós, Esther; Serrano, Diego; Puig-Kröger, Amaya; Riol, Lorena; Lasala, Fátima; Martinez, Iñigo; Vidal-Vanaclocha, Fernando; Delgado, Rafael; Rodríguez-Fernández, José Luis; Rivas, Luis; Corbí, Angel L.; Colmenares, María title: Role of the C-type lectins DC-SIGN and L-SIGN in Leishmania interaction with host phagocytes date: 2005-08-19 journal: Immunobiology DOI: 10.1016/j.imbio.2005.05.013 sha: doc_id: 322617 cord_uid: znvikfza Leishmaniasis is a parasitic disease that courses with cutaneous or visceral clinical manifestations. The amastigote stage of the parasite infects phagocytes and modulates the effector function of the host cells. Our group has described that the interaction between Leishmania and immature monocyte-derived dendritic cells (DCs) takes place through dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a C-type lectin that specifically recognizes fungal, viral and bacterial pathogens. The DC-SIGN-mediated recognition of Leishmania amastigotes does not induce DC maturation, and the DC-SIGN ligand/s on Leishmania parasites is/are still unknown. We have also found that the DC-SIGN-related molecule L-SIGN, specifically expressed in lymph node and liver sinusoidal endothelial cells, acts as a receptor for L. infantum, the parasite responsible for visceral leishmaniasis, but does not recognize L. pifanoi, which causes the cutaneous form of the disease. Therefore, DC-SIGN and L-SIGN differ in their ability to interact with Leishmania species responsible for either visceral or cutaneous leishmaniasis. A deeper knowledge of the parasite-C-type lectin interaction may be helpful for the design of new DC-based therapeutic vaccines against Leishmania infections. vector, while L. infantum causes VL due to parasite dissemination into internal organs. Leishmania parasites exist in two developmental stages. The flagellated promastigote is transmitted with the bite of the sand fly (insect vector) to the mammalian host, where it transforms into the amastigote stage. Leishmania amastigotes infect mononuclear phagocytes, where their intracellular location allows them to subvert the effector and regulatory functions of these cells (Duclos and Desjardins, 2000) . Since epidermal Langerhans cells and dermal dendritic cells (DCs) contribute to immunosurveillance of the skin (Banchereau and Steinman, 1998; Mellman and Steinman, 2001) and are located in proximity to the site of parasite delivery, their role in the initiation of Leishmania-specific immune responses is an active area of research. To attain a successful infection, Leishmania needs to subvert the host immune response from the early steps after its inoculation. In the natural course of infection, these events occur in the dermis, where DCs may act as host cells for Leishmania, independently of the pathological outcome of the infection (McDowell et al., 2002) . In this regard, Langerhans cells within cutaneous lesions are parasitized by Leishmania in vivo in both human and experimental murine CL (Blank et al., 1993; Moll, 1993) . Studies on the interactions of Leishmania with murine or human DC have not yet clearly determined the range of parasite forms that these cells can internalize (Amprey et al., 2004; Bennett et al., 2001; Blank et al., 1993; Konecny et al., 1999; Marovich et al., 2000; Moll, 2000; Moll and Flohe, 1997; Qi et al., 2001; Sacks and Sher, 2002; Udey et al., 2001; von Stebut et al., 1998 von Stebut et al., , 2000 or their influence on parasite survival. Although Leishmania species might differentially subvert DC effector function (Antoine et al., 2004; Brandonisio et al., 2004; Chaussabel et al., 2003; Ghosh and Bandyopadhyay, 2004; Jebbari et al., 2002; Konecny, et al., 1999; Ponte-Sucre et al., 2001; Scott and Hunter, 2002; von Stebut et al., 1998) , the receptors involved in the Leishmania-DC interaction remain largely undefined and could be critical for this process. In contrast, several macrophage receptors have been identified which mediate binding and subsequent uptake of Leishmania promastigotes (Blackwell, 1985; Russell and Talamas-Rohana, 1989) . In this regard, lipophosphoglycan (LPG) and the metalloproteinase gp63 bind to complement receptor type 3 (CR3), mannose-fucose, and fibronectin receptors on macrophages (Blackwell, 1985; Da Silva et al., 1989; Guy and Belosevic, 1993; Mosser, 1994; Talamas-Rohana et al., 1990; Wilson and Pearson, 1988) . However, the receptors implicated in amastigote uptake by DC and macrophages are poorly characterized, mainly due to the fact that isolation or in vitro culture of this intracellular form is difficult. The availability of axenic cultures has now opened the possibility of addressing the identification of Leishmania amastigotes receptors on macrophages and DCs (Armson et al., 1999; Bates et al., 1992; Debrabant et al., 2004; Doyle et al., 1991; Gupta et al., 2001; Hodgkinson et al., 1996) . Macrophages and DCs express a wide variety of pathogen-associated molecular pattern receptors, including numerous C-type lectin and lectin-like receptors (Engering et al., 2002; Figdor et al., 2002; McGreal et al., 2004) . Since Leishmania spp. display an abundance of mannose-rich glycoconjugates on their surface that are important for parasite virulence (Ilgoutz and McConville, 2001; Garami and Ilg, 2001) , a reasonable hypothesis is that lectin-oligosaccharide interactions are involved in parasite recognition by mononuclear phagocytes. DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN, CD209) is a type II transmembrane C-type lectin expressed on DCs and macrophages, and was initially described as involved in cell-cell interactions through its capacity to bind ICAM-3 and ICAM-2 (Geijtenbeek et al., 2000a, b; van Kooyk and Geijtenbeek, 2002) . The DC-SIGN extracellular domain comprises eight 23-residue tandem repeats and a Cterminal carbohydrate-recognition domain (Mitchell et al., 2001) . DC-SIGN is now known to be a receptor for HIV (Geijtenbeek et al., 2000c; Pohlmann et al., 2001) , Ebola virus (Alvarez et al., 2002) , Schistosoma mansoni , Sindbis virus (Klimstra et al., 2003) Candida albicans (Cambi et al., 2003) Mycobacterium tuberculosis , Hepatitis C (Wang et al., 2004) , Helicobacter pylori (Bergman et al., 2004) and the fungal pathogen Aspergillus fumigatus . Most Leishmania amastigote-DC studies have been carried out with tissue-derived opsonized parasites, which might be bound via Fc and complement receptors, thus precluding the identification of opsonization-independent binding mechanisms. To analyze the participation of the receptor DC-SIGN in binding and internalization of Leishmania, we used axenic amastigotes (Armson et al., 1999; Pan and McMahon-Pratt, 1988) , which are devoid of opsonizing antibodies. We first analyzed the interaction of L. pifanoi (that causes CL) axenic amastigote with K562-DC-SIGN transfectants and demonstrated that they are specifically recognized by DC-SIGN (Colmenares et al., 2002) . Subsequently, L. pifanoi amastigotes were found to bind DC-SIGN on the surface of immature monocytederived dendritic cells (IMDDCs), an interaction that was dramatically reduced in the presence of anti-DC-SIGN blocking antibodies (Colmenares et al., 2002) . This set of results suggested an important role for DC-SIGN in the early stages of infection of DCs by Leishmania. Since infection of immature dermal DCs is a common step shared by all Leishmania species, we next analyzed the capacity of IMDDCs to bind other Leishmania life cycle forms and species. Our results underscored the relevance of the DC-SIGN-Leishmania interaction in both VL (L. infantum) and CL (L. pifanoi), as amastigotes and promastigotes from both species exhibited DC-SIGN-interaction capacity. Since the membrane composition of the parasite changes throughout its life cycle (Bahr et al., 1993; Wright and el Amin, 1989) , we tested the ability of DC-SIGN to bind the three main life cycle forms of the parasite (amastigotes, procyclic promastigotes and metacyclic promastigotes). Amastigotes and metacyclic promastigotes showed the strongest DC-SIGN-dependent interaction with IMDDC ( Fig. 1) . Moreover, the avidity for DC-SIGN increased in the transition from procyclic (non-infective) to metacyclic (infective) promastigotes (i.e., procyclic and metacyclic promastigotes, respectively) (Fig. 1) . Hence, the avidity of the different forms of the parasite for DC-SIGN appears to correlate with their virulence. On the other hand, a much lower ability for DC-SIGN recognition was observed in Leishmania major promastigotes (Fig. 1) . LPG is one of the most abundant glycoconjugates exposed on the cell surface of promastigotes, but scarcely expressed on amastigotes, and plays a pleiotropic role through the life cycle of Leishmania (Aebischer et al., 2005; Cunningham, 2002; Kamhawi et al., 2000; Naderer et al., 2004; Sacks et al., 2000; Turco and Descoteaux, 1992; Turco et al., 2001) . This molecule is characterized by a high mannose content, and has been proposed to mediate promastigote interaction with DCs via DC-SIGN . However, our results indicate that LPG is not an important Leishmania ligand for DC-SIGN because: (1) LPG is strongly down-regulated in amastigotes (Ilg et al., 1999; Ilgoutz and McConville, 2001; Turco and Descoteaux, 1992) , which exhibit the highest DC-SIGN-binding ability; (2) LPG was unable to block Leishmania binding to DC-SIGN for all species and parasite developmental stages assayed; and (3) the LPG-defective L. donovani promastigotes (R2D2) bind to DC-SIGN-expressing cells. Furthermore, since LPG-defective promastigotes bound DC-SIGN with higher avidity than their wild-type counterparts, LPG might in fact mask other promastigote membrane ligands with affinity for DC-SIGN . Leishmania ability to modulate the DC maturation state: Conflicting results have been obtained on the ability of various Leishmania species to induce DC maturation (Antoine et al., 2004) . It is currently unknown whether this variability is due to variations in the experimental conditions or truly reflects speciesspecific or strain-specific interactions between Leishmania and DC. Since Leishmania spp. express highly polymorphic cell surface molecules, the DC response might vary according to the Leishmania species examined (Bennett et al., 2001; Flohe et al., 1998; Henri et al., 2002; Konecny et al., 1999; McDowell et al., 2002; von Stebut et al., 1998 von Stebut et al., , 2000 . We have evaluated the ability of L. infantum to alter the maturation state of IMDDC. Unlike LPS, L. infantum amastigotes, which bind to IMDDC via DC-SIGN, did not affect the cell surface expression of CD83, CD86 or MHC II, commonly considered as DC maturation parameters. Therefore, our results suggest that non-opsonized L. infantum amastigotes are unable to induce IMDDC maturation, at least during a 48-hour period ( Fig. 2A) . Moreover, LPS induced maturation of IMDDCs infected with L. infantum amastigotes ( Fig. 2A) , indicating that the parasites do not inhibit the capacity of DCs to be matured by other pathogen-derived products. The failure of IMDDC to mature in response to Leishmania capture and entry might represent a parasite strategy to avoid immunosurveillance and to allow their establishment and multiplication before the onset of immune responses. On the other hand, L. infantum amastigotes did not induce CCR7-directed IMDDC migration but Immature monocyte-derived DCs were prepared as previously described, left untreated or pre-treated 10 min at 25 1C with the blocking anti-DC-SIGN monoclonal antibody (Relloso et al., 2002) and incubated with CFSE-labeled parasites at a 1:5 ratio for 1 h at 35 1C. Afterward, the percentage of cells with bound parasites was quantified by flow cytometry and the contribution of DC-SIGN (%) was calculated as: 100À((% MR-1treated cells with bound parasites  100)/% untreated cell with bound parasites). inhibited the CCR7-dependent migration induced upon LPS maturation (Fig. 2B) . These data are in agreement with previous results demonstrating that cytokines abundantly produced during Leishmania infection (e.g., IL-10) down-regulate CCR7 expression (Antoine et al., 2004) . Therefore, Leishmania might prevent the establishment of T cell-mediated immunity by interfering with the migratory properties of DCs. L-SIGN: a receptor implicated in VL: L-SIGN is a close homologue of DC-SIGN (77% amino acid sequence identity) that is expressed on human liver and lymph node sinusoidal endothelial cells (Soilleux et al., 2000) . Like DC-SIGN, L-SIGN recognizes and binds high-mannose glycans, binds to ICAM-3 (Bashirova et al., 2001) , and recognizes carbohydrate structures on pathogens such as ManLAM on M. tuberculosis (Koppel et al., 2004) and high-mannose moieties on HIV-1, HCV and Ebola. Besides, L-SIGN has been described as a receptor for severe acute respiratory syndrome coronavirus (Jeffers et al., 2004) . Unlike DC-SIGN, L-SIGN does not bind to the fucose-containing Lewis x antigens, suggesting that L-SIGN-expressing liver endothelial cells and lymph node are not involved in capture and internalization of Lewis x -containing pathogens such as H. pylori and S. mansoni (Van Liempt et al., 2004) . Besides, binding to L-SIGN is not reversible at low pH, suggesting that L-SIGN does not release internalized ligand in low-pH vesicles and that L-SIGN is degraded upon internalization (Guo et al., 2004) . Because of their similar ligand specificity but differential tissue location, we have compared the capacity of DC-SIGN and L-SIGN to bind axenic amastigotes from L. pifanoi (responsible for CL), and L. infantum (responsible for VL). Binding experiments with Jurkat cells stably transfected with DC-SIGN or L-SIGN indicated that L. infantum amastigotes specifically bound to both DC-SIGN and L-SIGN, whereas L. pifanoi amastigotes were unable to bind to L-SIGN (Fig. 3) . Therefore, only VL-causing parasites (L. infantum) appear to be recognized by L-SIGN. These results suggest that L-SIGN recognition of the distinct Leishmania species might play a role in the outcome of the parasite infection (CL vs. VL). To further evaluate the relevance of L-SIGN in binding of Leishmania species causing VL, human hepatic sinusoidal endothelial cells (HSEC) were isolated from hepatic surgery donors, using a modification (In˜igo Martinez and Fernando Vidal-Vanaclocha, unpublished information) of previously described isolation procedures (Daneker et al., 1998; Heuff et al., 1994) . Incubation of HSEC with axenic amastigotes showed that L. infantum amastigotes bound strongly to HSEC, whereas no binding was observed with L. pifanoi amastigotes (Fig. 4) . In addition, the HSEC-L. infantum (Alvarez et al., 2002) were left untreated or pretreated with receptor-specific blocking antibodies, and then incubated at 35 1C with the indicated CFSE-labeled parasites (1:5 cell:parasite ratio), or left uninfected (Colmenares et al., 2002) . The percentage of cells with bound parasites was quantified by flow cytometry. Three independent experiments were performed with similar results, and a representative experiment is shown. amastigote interaction was reduced in the presence of a blocking monoclonal antibody against L-SIGN (Fig. 4) . These results confirmed the ability of VL-causing amastigotes to interact with L-SIGN. Given the mechanism described for the hepatitis C virus (Cormier et al., 2004) , it is tempting to speculate that L-SIGNmediated capture of L. infantum by HSEC could result in transinfection of Kupffer cells, which are the final targets of VL-causing Leishmania parasites (el Hag et al., 1994; Murray, 2001) . Taken together, the present work demonstrates that the C-type lectins DC-SIGN and L-SIGN are broad Leishmania receptors that differentially bind the distinct infective forms and species of the parasite. A deeper knowledge of the Leishmania-DC/L-SIGN interactions, and the subsequent immune consequences, may pave the way for the design of new therapeutic approaches against leishmaniasis. The recent description that macrophage treated with IL-4 (alternatively activated macrophages) also express DC-SIGN (Puig-Kroger et al., 2004) could be of major importance, due to the fact that the expression of this cytokine correlates with the pathology of the disease, and hence increases the potential relevance of these C-type lectins in leishmaniasis. Fig. 4 . L. infantum binding to hepatic sinusoidal endothelial cells (HSEC) is partially mediated by L-SIGN. Human HSEC were isolated from liver biopsies, and subjected to binding assays with L. pifanoi or L. infantum axenic amastigotes, as described (Colmenares et al., 2002) . After incubation and washing, cells were photographed (HSEC, hepatic sinusoidal endothelial cells; K, Kupffer cells). Two independent experiments were performed with similar results, and a representative experiment is shown. A critical role for lipophosphoglycan in proinflammatory responses of dendritic cells to Leishmania mexicana C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans Inhibition of CD1 expression in human dendritic cells during intracellular infection with Leishmania donovani Leishmania spp.: on the interactions they establish with antigen-presenting cells of their mammalian hosts Cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells A comparison of the effects of a benzimidazole and the dinitroanilines against Leishmania infantum Expression of lipophosphoglycan, highmolecular weight phosphoglycan and glycoprotein 63 in promastigotes and amastigotes of Leishmania mexicana Dendritic cells and the control of immunity A dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes HIV-1 infection Axenic cultivation and characterization of Leishmania mexicana amastigote-like forms Silent infection of bone marrowderived dendritic cells by Leishmania mexicana amastigotes /T helper cell 2 balance through phase-variable interaction between lipopolysaccharide and DC-SIGN Receptors and recognition mechanisms of Leishmania species Parasitism of epidermal Langerhans cells in experimental cutaneous leishmaniasis with Leishmania major Dendritic cells in Leishmania infection The C-type lectin DC-SIGN (CD209) is an antigen-uptake receptor for Candida albicans on dendritic cells Unique gene expression profiles of human macrophages and dendritic cells to phylogenetically distinct parasites Dendritic cell (DC)-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN, CD209), a C-type surface lectin in human DCs, is a receptor for Leishmania amastigotes The dendritic cell receptor DC-SIGN discriminates among species and life cycle forms of Leishmania L-SIGN (CD209L) and DC-SIGN (CD209) mediate transinfection of liver cells by hepatitis C virus Parasitic adaptive mechanisms in infection by leishmania CR1, the C3b receptor, mediates binding of infective Leishmania major metacyclic promastigotes to human macrophages Culture and characterization of sinusoidal endothelial cells isolated from human liver Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics Leishmania donovani: long-term culture of axenic amastigotes at 37 degrees C Subversion of a young phagosome: the survival strategies of intracellular pathogens Liver morphology and function in visceral leishmaniasis (Kala-azar) Immune escape through C-type lectins on dendritic cells C-type lectin receptors on dendritic cells and Langerhans cells Antigenpulsed epidermal Langerhans cells protect susceptible mice from infection with the intracellular parasite Leishmania major Disruption of mannose activation in Leishmania mexicana: GDP-mannose pyrophosphorylase is required for virulence, but not for viability DC-SIGN-ICAM-2 interaction mediates dendritic cell trafficking Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses DC-SIGN, a dendritic cellspecific HIV-1-binding protein that enhances trans-infection of T cells Mycobacteria target DC-SIGN to suppress dendritic cell function Interaction of Leishmania parasites with dendritic cells and its functional consequences Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR In vitro cultivation and characterization of axenic amastigotes of Leishmania Comparison of receptors required for entry of Leishmania major amastigotes into macrophages Hierarchy of susceptibility of dendritic cell subsets to infection by Leishmania major: inverse relationship to interleukin-12 production Isolation of rat and human Kupffer cells by a modified enzymatic assay Leishmania amazonensis: cultivation and characterization of axenic amastigote-like organisms Proteophosphoglycans from Leishmania promastigotes and amastigotes Function and assembly of the Leishmania surface coat Leishmania major promastigotes inhibit dendritic cell motility in vitro CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus The vectorial competence of Phlebotomus sergenti is specific for Leishmania tropica and is controlled by species-specific, lipophosphoglycan-mediated midgut attachment DC-SIGN and L-SIGN can act as attachment receptors for alphaviruses and distinguish between mosquito cell-and mammalian cell-derived viruses Murine dendritic cells internalize Leishmania major promastigotes, produce IL-12 p40 and stimulate primary T cell proliferation in vitro Identification of the mycobacterial carbohydrate structure that binds the C-type lectins DC-SIGN, L-SIGN and SIGNR1 IL-12p70 production by Leishmania majorharboring human dendritic cells is a CD40/CD40 liganddependent process Leishmania priming of human dendritic cells for CD40 ligand-induced interleukin-12p70 secretion is strain and species dependent Divergent roles for C-type lectins expressed by cells of the innate immune system Dendritic cells: specialized and regulated antigen processing machines A novel mechanism of carbohydrate recognition by the C-type lectins DC-SIGN and DC-SIGNR. Subunit organization and binding to multivalent ligands Experimental cutaneous leishmaniasis: Langerhans cells internalize Leishmania major and induce an antigen-specific T-cell response The role of dendritic cells at the early stages of Leishmania infection Dendritic cells induce immunity to cutaneous leishmaniasis in mice Receptors on phagocytic cells involved in microbial recognition Tissue granuloma structure-function in experimental visceral leishmaniasis Surface determinants of Leishmania parasites and their role in infectivity in the mammalian host Monoclonal antibodies specific for the amastigote stage of Leishmania pifanoi. I. Characterization of antigens associated with stage-and species-specific determinants DC-SIGNR, a DC-SIGN homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans Leishmania major lipophosphoglycan modulates the phenotype and inhibits migration of murine Langerhans cells ICAM-3)-grabbing nonintegrin in THP-1 human leukemic cells, monocytes, and macrophages Leishmania amazonensisdendritic cell interactions in vitro and the priming of parasite-specific CD4(+) T cells in vivo DC-SIGN (CD209) expression is IL-4 dependent and is negatively regulated by IFN, TGF-beta, and anti-inflammatory agents Leishmania and the macrophage: a marriage of inconvenience Evasion of innate immunity by parasitic protozoa The role of phosphoglycans in Leishmania-sand fly interactions Dendritic cells and immunity to leishmaniasis and toxoplasmosis Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin mediates binding and internalization of Aspergillus fumigatus conidia by dendritic cells and macrophages DC-SIGN; a related gene, DC-SIGNR; and CD23 form a cluster on 19p13 Lipophosphoglycan from Leishmania mexicana promastigotes binds to members of the CR3, p150,95 and LFA-1 family of leukocyte integrins The lipophosphoglycan of Leishmania parasites Is lipophosphoglycan a virulence factor? A surprising diversity between Leishmania species Skin dendritic cells in murine cutaneous leishmaniasis The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x A novel adhesion pathway that regulates dendritic cell trafficking and T cell interactions Molecular basis of the differences in binding properties of the highly related C-type lectins DC-SIGN and L-SIGN to Lewis X trisaccharide and Schistosoma mansoni egg antigens Uptake of Leishmania major amastigotes results in activation and interleukin 12 release from murine skin-derived dendritic cells: implications for the initiation of anti-Leishmania immunity Leishmania major-infected murine langerhans cell-like dendritic cells from susceptible mice release IL-12 after infection and vaccinate against experimental cutaneous Leishmaniasis DC-SIGN: binding receptors for hepatitis C virus Roles of CR3 and mannose receptors in the attachment and ingestion of Leishmania donovani by human mononuclear phagocytes Leishmania infection: surfaces and immunity This work was supported by the Ministerio de Educacio´n y Ciencia (Grants SAF2002-04615-C02-01, GEN2003-20649-C06-01/NAC and AGL2004-02148-ALI) and Fundacio´n para la Investigacio´n y Prevencio´n del SIDA en Espan˜a (FIPSE 36422/03) to ALC. MC was supported by the Ministerio de Educacio´n y Ciencia (Ramo´n y Cajal programme). EC was supported by a Fellowship FPI from the Ministerio de Educacio´n y Ciencia.