Editor: Willem van Eden
Leptospira interrogans is recognized through DC-SIGN and induces maturation and cytokine production by human dendritic cells
Article first published online: 28 JUN 2008
© 2008 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
FEMS Immunology & Medical Microbiology
Volume 53, Issue 3, pages 359–367, August 2008
How to Cite
Gaudart, N., Ekpo, P., Pattanapanyasat, K., Van Kooyk, Y. and Engering, A. (2008), Leptospira interrogans is recognized through DC-SIGN and induces maturation and cytokine production by human dendritic cells. FEMS Immunology & Medical Microbiology, 53: 359–367. doi: 10.1111/j.1574-695X.2008.00437.x
- Issue published online: 29 JUL 2008
- Article first published online: 28 JUN 2008
- Received 30 January 2008; revised 21 April 2008; accepted 22 April 2008.First published online 18 June 2008.
- Leptospira interrogans;
- monocyte-derived dendritic cell;
Leptospirosis is a global zoonotic disease, caused by pathogenic Leptospira species including Leptospira interrogans, that causes public health and livestock problems. Pathogenesis, immune response and cellular receptors for Leptospira are not well understood. Interaction of dendritic cells (DCs) with L. interrogans serovar Autumnalis L-643 and BL-6 isolated from leptospirosis patients, and both virulent and avirulent serovar Pyrogenes 2317 strains isolated from animal were investigated. Carbohydrate analysis using lectins showed that all of these leptospires contained high mannose components as a common backbone and DC-SIGN was involved in leptospires' attachment. Interaction of the L. interrogans strains with DCs induced maturation, but had different effects on IL-10, IL-12p70 and tumor necrosis factor (TNF)-α production. Both virulent and avirulent Pyrogenes 2317 and Autumnalis BL-6 but not L-643 strains induced IL-12p70 and TNF-α production, but minimal IL-10 secretion. These data demonstrated that L. interrogans binds DC-SIGN and induces DCs maturation and cytokine production, which should provide new insights into cellular immune processes during leptospirosis.
Leptospirosis is a global zoonotic disease caused by pathogenic species of Leptospira, including Leptospira interrogans (Brenner et al., 1999; Faine et al., 1999). The main route for Leptospira infection is via skin abrasions and through mucous membranes, resulting in various clinical symptoms ranging from flu-like syndromes to fatal illness (Levett, 2001). Virulent Leptospira strains may lose their virulence in long-term culture. However, the mechanism for this switch to avirulence is unknown (Faine et al., 1999).
Knowledge of host immunity to Leptospira and leptospirosis is limited. Leptospirosis is dependent on both host humoral and cell-mediated immunity (Faine et al., 1999). Th1-type immune response and γδT cells induced by peripheral blood mononuclear cells (PBMC) may play an important role in host defense against Leptospira in vitro and in animal models (Naiman et al., 2001, 2002; Klimpel et al., 2003). In addition, heat-killed pathogenic L. interrogans induces Th1 responses via secretion of tumor necrosis factor (TNF)-α, IFN-γ and IL-12p40 (de Fost et al., 2003). In leptospirosis patients, elevated levels of TNF-α but not IL-10 are associated with severity of disease and mortality (Estavoyer et al., 1991; Tajiki & Salomao, 1996).
Dendritic cells (DCs) are professional antigen-presenting cells that form an important link between innate and adaptive immune responses (Banchereau et al., 2000). Immature DCs reside in almost all tissues throughout the body, including the skin, where they can capture and process antigens. Next, DCs migrate to lymph nodes and present the processed antigens to naïve T cells. Depending on the pathogen encountered, DCs can produce specific cytokines and surface proteins that play a key role in the differentiation of naïve T helper cells towards Th1 or Th2 cells, which regulate the cellular and humoral immune response, respectively (de Jong et al., 2002).
DCs express numerous pattern recognition receptors (PRR) that recognize conserved pathogen-associated molecular patterns (Figdor et al., 2002; Janeway & Medzhitov, 2002). One family of PRR consists of C-type lectins that recognize specific carbohydrates on pathogens and on self-antigens, and play a role in antigen uptake and cell–cell adhesion (Figdor et al., 2002). One of the C-type lectins expressed on DCs is DC-Specific ICAM-3-Grabbing Nonintegrin (DC-SIGN) or CD 209, which recognizes high-mannose glycans as well as fucose-containing antigens on a variety of viruses, bacteria, parasites and self-antigens (Geijtenbeek et al., 2002; Geijtenbeek & van Kooyk, 2003). DC-SIGN has many functions on DC for activation of immune responses.
Knowledge of host cell receptors and immune responses to Leptospira remains limited and, in particular, reports of the interaction between Leptospira and DCs are lacking. As Leptospira are known to expose carbohydrates on their surface, this study examined the involvement of DC-SIGN as a recognition receptor for Leptospira. Also, we studied the consequences on DC function of interaction with Leptospira.
Materials and methods
Bacterial strains and culture
Leptospira interrogans serovar Autumnalis L-643 and BL-6 strains were isolated from a patient who died from leptospirosis and from a patient who recovered from leptospirosis after treatment, respectively. These isolates were collected from Regional Hospital, Loi province, Thailand, and passaged <10 times before use. Leptospira interrogans serovar Pyrogenes 2317 was collected from a wild rat and subsequently cultured until it became avirulent. These strains were kindly provided by the Department of Veterinary Medicine, Royal Thai Army-Armed Forces Research Institute for Medical Science (AFRIMS). The virulence of these stains was tested by injection of 0.5 × 108 cells mL−1 leptospires into a group of 4-week-old specific pathogen-free hamsters (4 SPF/group) and clinical symptoms were observed. Injection of the virulent strain resulted in death after 5 days whereas the avirulent strain did not induce disease. The serogroup of these strains was confirmed by serotyping by WHO, Australia. The reference L. interrogans and Leptospira biflexa (Table 1) were kindly provided by the Reference Collection of the National Institute of Animal Health (NIAH), Department of Livestock Development, Ministry of Agricultural and Cooperatives, Thailand, and by the National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Thailand. Leptospires were grown in liquid Ellinghausen McCullough–Johnson–Harris (EMJH) medium supplemented with 3% rabbit serum at 30 °C (Ellinghausen & McCullough, 1965) to a density of about 108 bacteria mL−1. These reference leptospires were washed three times with phosphate-buffered saline (PBS) pH 7.2 to eliminate rabbit serum background and killed with 2% paraformaldehyde before use as whole-cell antigens. Salmonella typhimurium ATCC 131 lot 841 was cultured in LB broth.
|Pyrogenes 2317 vi||+++||−||−||++||−||−||++|
|Pyrogenes 2317 avi||+++||+||−||+++||+||−||+++|
The following biotinylated lectins (Vector Laboratories) with defined carbohydrate specificities were used: concanavalin A (ConA), Dolichos biflorus (DBA), peanut agglutinin (PNA), Ricinus communis agglutinin (RCA120), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA1). Killed leptospires (107 cells) were coated onto a Maxisorp plate (Nunc), biotinylated lectin was added and binding was detected with alkaline phosphatase-conjugated streptavidin (Southern Biotechnology).
Soluble DC-SIGN-Fc adhesion assay
Binding to soluble DC-SIGN was analyzed using DC-SIGN-Fc in an enzyme-linked immunosorbent assay (ELISA)-based assay as described previously (Geijtenbeek et al., 2000a, b, c). In brief, killed leptospires (107 cells) were coated onto a Maxisorp plate, and subsequently, wells were treated with 5% skimmed milk, incubated with soluble DC-SIGN-Fc and binding was detected with peroxidase-conjugated goat anti-mouse immunoglobulin. Where indicated, DC-SIGN-Fc was preincubated with 50 μg mL−1 mannan (Sigma) for 20 min at room temperature. Mannan (100 ng mL−1) was coated and used as a positive control whereas buffer was used as a negative control.
Human myelogenous leukemia K-562 cells and K-562 cells transfected with DC-SIGN (K-SIGN) were used. K-562 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS, BioWithaker), glutamine and penicillin–streptomycin. K-SIGN cells were cultured in a mixture of 75% RPMI 1640, 25% ISCOV's Dulbecco's Modified Eagle's Medium (DMEM) (both from Gibco) supplemented with 10% FBS and 2 mg mL−1 G418 (Gibco) as described previously (Geijtenbeek et al., 2000a, b, c). Peripheral blood was obtained from healthy members of the Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University. PBMC were isolated by centrifugation using Histopaque-1077 (Sigma). CD14+ monocytes were isolated using CD14 MACS MicroBeads (Miltenyi Biotec) according to the manufacturer's instructions. Monocyte-derived DCs (MoDCs) were generated by culturing monocytes for 5–7 days in RPMI 1640 with nonessential amino acids, 2 mM l-glutamine, 1 mM sodium pyruvate, 100 μg mL−1 penicillin and 100 μg mL−1 streptomycin (all from Gibco) containing 10% FBS in the presence of 100 ng mL−1 rhIL-4 and 100 ng mL−1 recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) (both from R&D Systems). The level of DC-SIGN expression was determined by staining with mouse anti-DC-SIGN antibody (AZN-D1), followed by goat anti-mouse fluorescein isothiocyanate (FITC)-antibody and analyzed by flow cytometry (FACS Calibur, Becton Dickinson).
Bacteria (109 cells mL−1) were labeled with FITC (1 mg mL−1, Sigma) for 1 h at room temperature, washed and killed with 2% paraformaldehyde as described previously (Bergman et al., 2004). Labeled bacteria were added to cells (100 bacteria per cell) and incubated for 40 min at 37 °C. Binding was analyzed using flow cytometry. Where indicated, cells were pretreated with AZN-D1 (20 μg mL−1) (Geijtenbeek et al., 2000a, b, c), mannan (50 μg mL−1) or EDTA (10 mM) for 20 min at room temperature. Salmonella typhimurium was used as a positive control and medium was used as a negative control. Data are shown as percent positive cells.
Immature MoDCs were incubated with formaldehyde-killed Leptospira (MoDCs : Leptospira 1 : 100) or ultrapure Escherichia coli K12 lipopolysaccharide (5 μg mL−1, InvivoGen). After 2 days, DCs were harvested, stained for cell-surface expression of CD83 and CD86 with anti CD83-FITC and anti-CD86-PE antibodies (both from BD Bioscience) and analyzed using flow cytometry. Culture supernatant was collected for quantification of IL-10, IL-12p70 and TNF-α using cytokine ELISA kits according to the manufacturer's instructions (R&D Systems).
Statistical analysis was performed using microsoft excel and sigma plot software. The mean values of experiments were compared using Student's t-test. P<0.05 was considered to be significant.
Expression of carbohydrates on L. interrogans
We hypothesized that L. interrogans may contain carbohydrates that are potential binding sites for the C-type lectin DC-SIGN. Carbohydrates expressed were identified using biotinylated plant lectins with well-characterized carbohydrate-binding specificity. Whole cells of 22 reference pathogenic L. interrogans serovars, clinical isolates of serovar Autumnalis L-643 and BL-6 strains as well as animal isolates of serovar Pyrogenes 2317, both virulent and avirulent strains, and nonpathogenic L. biflexa serovar Patoc I were investigated. All tested leptospires reacted strongly to ConA, specific for high mannose-containing carbohydrate, and to varying extents with the lectin RCA120 and WGA, specific for Galβ1–4GlcNAcβ1–R and GlcNAcβ1–4GlcNAcβ1–4GlcNAc, respectively, but not or weakly to the other lectins (Table 1). Although all serovars reacted with ConA and thus contain high mannose, the configuration and spacing of high mannose may still differ between these divergent serovars. As DC-SIGN has a carbohydrate specificity for carbohydrates with high mannose components, these results indicated that all reference strains and clinical isolates contain carbohydrates that can potentially interact with DC-SIGN.
Interaction of DC-SIGN with L. interrogans
Next, we determined whether indeed the L. interrogans species could interact with DC-SIGN as predicted from the presence of high mannose carbohydrate on their cell surface. For this, we used a cell-free assay in which binding of soluble DC-SIGN molecules coupled to the Fc portion of human IgG1 was evaluated in an ELISA-based binding assay (Geijtenbeek et al., 2000a, b, c). Clinical isolates L. interrogans serovars Autumnalis, Bataviae, Javanica and Sejroe, which were the predominant serovars isolated in Thailand from 2000 to 2004 (Wuthiekanun et al., 2007), serovars Autumnalis L-643 and BL-6 strains and serovar Pyrogenes 2317 virulent (vi) strain and its corresponding avirulent (avi) strain could specifically bind soluble DC-SIGN-Fc, although the levels of binding varied among the serovars (Fig. 1). Serovar Autumnalis L-643, but not Autumnalis BL-6, and both virulent and avirulent Pyrogenes 2317 showed high binding to DC-SIGN-Fc. This binding was specific as indicated by abrogation of binding using mannan as a competitor ligand. The DC-SIGN-binding component was confirmed by comparing between reference leptospires' whole cells and their corresponding secreted antigens. These secreted antigens contained carbohydrate components as identified by treatment with either sodium periodate or proteinase K, sodium dodecyl sulfate polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue and silver nitrate (data not shown). The results showed that whole-cell leptospires reacted to soluble DC-SIGN-Fc about 1.6–1.8-fold higher than their corresponding secreted antigens (data not shown). This indicated that L. interrogans contains carbohydrate components that can bind DC-SIGN. Thus, L. interrogans serovars have an ability to bind to soluble DC-SIGN.
Next, binding was confirmed using cellular DC-SIGN, on K-562 cells transfected with DC-SIGN (K-SIGN), and on MoDCs. As shown in Fig. 2a, both K-SIGN and MoDCs expressed DC-SIGN. We selected Autumnalis and Pyrogenes strains, based on their high affinity for DC-SIGN-Fc, for further studies. FITC-labeled leptospires were added to these cells at a ratio of 100 bacteria per cell for 40 min at 37 °C and binding was analyzed using flow cytometry. For all serovars tested, L. interrogans could bind to K562 cells, but higher levels of binding were obtained using K-SIGN (Fig. 2b). Binding was reduced to levels observed with K562 using mannan as a competitor ligand, EDTA to chelate Ca2+ that is required for ligand binding to C-type lectins, or anti-DC-SIGN antibodies, indicating that binding was specific for DC-SIGN (Fig. 2b). All serovars bound K-SIGN at similar levels, in contrast to the results using soluble DC-SIGN, which showed reduced affinity of Autumnalis BL-6. Compared with K-SIGN, all serovars could bind to MoDCs at much higher levels (Fig. 2c, note the scale difference). Interestingly, whereas binding of Autumnalis L-643 and BL-6 strains was for the larger part mediated by DC-SIGN (decrease of binding of 86% and 51%, respectively, using anti-DC-SIGN antibodies), binding of Pyrogenes strains was only partially attributed to DC-SIGN (virulent, 35% decrease in binding; avirulent, 42% decrease). Mannan and EDTA could only partially inhibit binding of certain serovars [Autumnalis BL-6 and Pyrogenes (vi) strains] as compared with anti-DC-SIGN antibodies. However, it should be noted that these compounds have a much broader action and could potentially inhibit additional C-type lectin receptors, such as a mannose receptor. As compared with L-643, Autumnalis BL-6 showed low affinity to DC-SIGN-Fc, lower binding to K-SIGN and binding to DCs could only be blocked partially using EDTA and mannan. Nevertheless, anti-DC-SIGN antibodies could reduce binding of BL-6 to DCs to background levels (Fig. 2c and consistent observations from four additional donors, data not shown), indicating that indeed DC-SIGN functions as a receptor for Autumnalis BL-6 on DCs. Interestingly, avirulent Pyrogenes showed higher binding to DCs and also higher affinity for DC-SIGN compared with virulent Pyrogenes, using both soluble as well as cellular DC-SIGN on transfected cells and MoDCs. Overall, these results showed that one of the receptors involved in binding L. interrogans is the C-type lectin DC-SIGN.
DCs activation induced by L. interrogans
Most pathogens that bind DCs induce phenotypical and functional changes. Therefore, we investigated the impact of L. interrogans on DC maturation and cytokine production using clinical isolates. MoDCs were cocultured with L. interrogans for 2 days; subsequently, the cells were stained for the maturation markers CD83 and CD86 and production of the cytokines IL-12p70, TNF-α and IL-10 as measured by ELISA. Escherichia coli lipopolysaccharide was used as a positive control for DC activation. As shown in Fig. 3, all L. interrogans strains induced DC maturation to a similar extent as assessed by the increased expression of CD83 and CD86. However, the profile of cytokine production was different. Autumnalis L-643 induced very low or undetectable levels of TNF-α and no IL-12p70 and IL-10 production, whereas MoDCs stimulated by Autumnalis BL-6 produced moderate levels of these cytokines (Fig. 4a–c). By contrast, both virulent and avirulent Pyrogenes induced high IL-12p70 and TNF-α production, but low IL-10 secretion. It should be noted that the levels of the anti-inflammatory cytokine IL-10 were low to moderate after stimulation with all serovars even in donors that were capable of IL-10 production upon activation by E. coli lipopolysaccharide.
In conclusion, all L. interrogans serovars could induce DC maturation but differed in the induction of cytokine production. Leptospira interrogans Pyrogenes strains, especially the virulent strain, induced higher IL-12p70 and TNF-α by MoDCs than Autumnalis strains. Also, we tested the ability of these Leptospira-activated MoDCs to polarize naïve T cell differentiation, and for all strains, no specific polarization into Th1 or Th2 cells was observed but rather a mixture of Th1/Th2 cells was obtained in most donors (data not shown).
The mechanisms that control the immune response to Leptospira are not completely understood. Most studies have used PBMC, monocytes or macrophages activated with Leptospira. Our results have provided the first demonstration of an interaction between L. interrogans and human MoDCs via C-type lectin DC-SIGN. Moreover, we showed that the interaction of L. interrogans with human MoDCs resulted in DC maturation and production of IL-12p70 and TNF-α.
Leptospira interrogans contain high mannose as the major surface carbohydrate as shown in this study using lectins with defined carbohydrate specificity. This may be related to the mannobiose unit, a major polysaccharide molecule on leptospiral lipopolysaccharide, which is an antigenic epitope and genus-specific antigen (Mutsuo et al., 2000). Leptospiral surface carbohydrates were recognized by both soluble DC-SIGN-Fc and cells expressing DC-SIGN, indicating that DC-SIGN is one of the receptors used by L. interrogans to interact with DCs. However, other receptors are involved as binding to DCs of Autumnalis strains was for the most part mediated by DC-SIGN but that of Pyrogenes strains could only be partially inhibited by anti-DC-SIGN antibodies. The CR3 receptor on MoDCs and the fibronectin-binding protein on L. interrogans might enhance the cellular adherence of pathogenic bacteria (Merien et al., 2000; Cinco et al., 2002). In addition to whole cells, secreted antigens from leptospires also interacted with DC-SIGN. These secreted antigens could potentially modulate the function of bystander DCs; however, the functional relevance of this interaction remains to be studied.
Leptospira interrogans serovars Autumnalis and Pyrogenes induced DC maturation and production of IL-12p70 and TNF-α. This is in line with other Spirochetes, such as Treponema palliduma and Borrelia burgdoferi, which can also induce DC maturation and cytokine production in vitro and in vivo (Bouis et al., 2001; Salazar et al., 2005; Sjowall et al., 2005). Although the mechanisms involved in DC activation were not studied here, the Toll-like receptor family of PRR are probable candidates (Netea et al., 2004). Recent reports showed that L. interrogans can activate immune responses by TLR-2 and TLR-4 (Werts et al., 2001; Nahori et al., 2005; Viriyakosol et al., 2006). TLR-2 acts as a receptor for outer membrane proteins such as LipL32 of pathogenic leptospires whereas TLR-4 activates immune responses through other undefined molecules. Because MoDCs express both TLR2 and TLR4, it is likely that these PRR play a role in the observed DCs' activation. Cross-talk between DC-SIGN and TLR signaling pathways during microbial sensing and recognition has been documented (Geijtenbeek et al., 2003; van Kooyk & Geijtenbeek, 2003), and this topic is of interest for future studies of leptospires and DCs.
The roles of TNF-α and IL-12, important cytokines with proinflammatory and antibacterial effects, in leptospirosis in humans are unknown. Chierakul et al. (2004) reported high plasma concentrations of IL-12p40 and TNF-α in Thai adult leptospirosis patients. However, it is unclear whether these cytokines are beneficial or worsen the outcome. We found that different L. interrogans strains induced different levels of IL12p70 and TNF-α production by DCs. The amount of cytokines produced was not linked to binding to DCs as L. interrogans Autumnalis strains bound to and activated MoDCs maturation similar to Pyrogenes virulent strains but they initiated lower or undetectable IL-12p70 and TNF-α secretion. On the other hand, avirulent L. interrogans Pyrogenes bound MoDCs to a higher extent than the virulent strain, but initiated less IL-12p70 and TNF-α secretion. In vivo and in vitro models showed that the infectivity of leptospires does not correlate with a loss of virulence because low passaged leptospires with a progressive loss of virulence and nonpathogenic strains can still infect target cells (Meiren et al., 1997). However, changes in surface components, including lipopolysaccharide, of virulent L. interrogans reported after in vitro cultivation might explain the observed differences between virulent and avirulent Pyrogenes (Haake et al., 1991; Meiren et al., 1997).
A high IL-10/TNF-α ratio is related to a good prognosis in human leptospirosis (Tajiki & Salomao, 1996). However, we observed only minor IL-10 production upon coculture of DCs with leptospires, even in donors that were capable of high IL-10 production by E. coli lipopolysaccharide. Interestingly, Autumnalis L-643 that was isolated from a blood sample of a deceased leptospirosis patient showed very low or undetectable levels of both TNF-α and IL-10 production by MoDCs. These results might suggest that DC-derived cytokines did not play a role in the mortality of this patient and other factors may involve cytokines produced by other cell types and/or the host immune status.
Polarization of the immune response depends on the balance between CLR and TLR activation (Geijtenbeek & van Kooyk, 2003). Several pathogens, such as Mycobacterium, Leishmania and Helicobacter, manipulate the balance of Th1/Th2 to cause chronic infections through CLR and TLR (Colmenares et al., 2002; Geijtenbeek et al., 2003; Bergman et al., 2004). We did not observe consistent skewing to Th1 or Th2 by Leptospira-activated DCs; however, there was a large donor variation and data were not conclusive.
In summary, the interaction of L. interrogans with DCs is in part mediated by DC-SIGN and might result in maturation and production of IL-12 and TNF-α. Further investigations on target host cell surface receptors, intracellular fate of L. interrogans within DCs and subsequent induction of T cell response are required. The results of such studies should provide new insights into the cellular immune processes of leptospirosis and should enhance our understanding of innate immunopathogenesis processes.
We thank Assoc. Prof. Yupin Suputtamongkol, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Col. Dr Duangporn Phulsuksombati and Major Noppadol Sangjun, Department of Veterinary Medicine, Royal Thai Army-AFRIMS, for providing Leptospira bacteria and for virulence testing in hamster; Dr Sathit Pichayangkul and Mr Kosol Yongvanitchit, Department of Immunology and Medicine, USAMC-AFRIMS, for their expert assistance in DCs culture and flow cytometry; and Dr Prapon Wilairat, Department of Biochemistry, Faculty of Science, Mahidol University, for assistance with the manuscript. N.G. is supported by a Thesis Grant, Faculty of Graduate Studies, and by Siriraj Graduate Thesis Scholarship, Faculty of Medicine Siriraj Hospital, Mahidol University. A.E. is supported by a grant from the Dutch Organization for Scientific Research (NWO grant no. 916.36.009).
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