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Originally published online as doi:10.1189/jlb.0706439 on January 29, 2007

Published online before print January 29, 2007
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(Journal of Leukocyte Biology. 2007;81:1188-1196.)
© 2007 by Society for Leukocyte Biology

The role of mannose receptor during experimental leishmaniasis

Oleg E. Akilov1,2, Rachel E. Kasuboski1, Cristina R. Carter and Mary Ann McDowell3

Center for Global Health and Infectious Diseases, Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana, USA

3 Correspondence: Department of Biological Science, University of Notre Dame, 215 Galvin Life Sciences, Notre Dame, IN 46556, USA. E-mail: mcdowell.11{at}nd.edu

ABSTRACT

The primary host cells for Leishmania replication are macrophages (MP). Several molecules on the surface of professional phagocytic cells have been implicated in the initial process of parasite internalization and initiation of signaling pathways. These pattern recognition receptors distinguish molecular patterns on pathogen surfaces. Mannose receptor (MR), specifically, recognizes mannose residues on the surface of Leishmania parasites. We studied the role of MR in the pathogenesis of experimental cutaneous and visceral leishmaniasis using MR-deficient [MR-knockout (KO)] C57BL/6 mice. MR-deficient MP exhibited a comparable infection rate and cytokine production. In the absence of MR, the clinical course of Leishmania major and Leishmania donovani infections was similar in MR-KO and wild-type mice (MR-WT). Furthermore, immunohistochemistry of cutaneous lesions from MR-KO and MR-WT mice revealed no differences in lesion architecture or cell components. Inhibition of MP responses is a hallmark of Leishmania infection; our data demonstrate further that host MR is not essential for blocking IFN-{gamma}/LPS-induced IL-12 production and MAPK activation by Leishmania. Thus, we conclude that the MR is not essential for host defense against Leishmania infection or regulation of IL-12 production.

Key Words: Leishmania • macrophages • pattern recognition

INTRODUCTION

Leishmaniasis is a vector-transmitted disease exhibiting a spectrum of clinical manifestations, ranging from localized, cutaneous lesions to fatal, visceral disease. These varied clinical outcomes are generally attributed to differences in the Leishmania species initiating the infections. For example, the Old World parasite Leishmania major induces cutaneous lesions, which heal spontaneously, referred to as cutaneous leishmaniasis (CL). The pathology associated with CL is immune-mediated and is marked by elimination of the parasites in the skin as a result of Th1-adapative immunity [1 ]. The most debilitating form of infection is visceral leishmaniasis (VL) or kala-azar. This disease is associated most often with infection with Leishmania donovani in the Old World and Leishmania infantum (=chagasi) in the New World. VL is a severe, chronic, systemic illness with multiorgan pathology and uncontrolled replication of Leishmania in the liver, spleen, and bone marrow. If the symptoms are left untreated, the fatality rate can reach up to 90% of cases.

Leishmania spp. have a digenetic life-cycle, alternating between free-living, flagellated, procyclic promastigotes in phlebotomine sand flies and obligate, intracellular, aflagellated amastigotes, which multiply within phagocytes of the vertebrate host. Leishmania do not invade host cells actively but rely on the phagocytic capacity of these cells to gain entry. This process is receptor-mediated [2 , 3 ], eventually resulting in the formation of a phagolysosome, and is known to induce host-cell signal transduction pathways [4 ]. Many receptors have been implicated in the entry of Leishmania parasites, including mannose receptor (MR) [5 , 6 ], complement receptors 1 and 3 (CR1/3) [7 , 8 ], FcR [9 ], and scavenger receptors [10 ].

MR is part of the innate system of "pattern recognition" to discriminate between infectious nonself and self. MR plays an important role during innate immune responses by recognition and phagocytosis of several microorganisms, such as mycobacteria [11 ] Candida albicans [12 ], Pneumocystis carinii [13 ], and Cryptococcus neoformans [14 ]. The evidence for MR involvement in the entry of Leishmania spp. has been varied and depends on the species and life-cycle stage used in the studies. Known MR ligands are able to partically block attachment and entry of L. donovani promastigotes into human monocyte-derived macrophage (MP) [6 , 15 ] and murine peritoneal MP [16 ], a phenomenon that has been inconsistent during in vitro infection with amastigotes [5 ]. Consistent with reports that little to no MR is expressed in human monocytes [17 , 18 ], blocking MR does not inhibit L. donovani attachment to these cells [15 ].

The primary focus on entry mechanisms of L. major has been on the role of CR3 [4 , 7 , 8 , 19 20 21 ], and few studies have addressed MR. Although it is clear that L. major amastigotes do not use MR for MP attachment [4 ], Palatnik et al. [22] observed an ~50% inhibition of L. major promastigote entry after preincubation with a MR ligand. The only other study to address L. major promastigote entry and MR has been in assessing epidermal Langherhans cells. No inhibition was observed in the presence of soluble mannan; however, it has been shown that these cells do not express MR [19 ].

MR is a member of the MR family, a subgroup of the C-type lectin superfamily, together with the M-type phospholipase A2 receptor, DEC-205/gp200-MR6 and Endo180/uPARAP [23 ], and is expressed mainly on cells of the myeloid lineage with only a few exceptions. MR recognizes branched polysaccharides with terminal mannose, fucose, or N-acetylglucosamine, which adorn the cell surfaces of a wide array of infectious agents as well as several endogenous ligands. The repetitive structure and glycan modifications associated with many Leishmania cell surface molecules suggest that these parasites interact with MR [24 ], which has not been shown to bind Leishmania molecules directly; however, the major surface glycoconjugate, lipophosphoglycan (LPG), of L. major and L. donovani promastigotes binds mannose-binding protein (MBP) [25 ]. It is interesting that MBP has the same binding specificity as the fourth carbohydrate recognition domain (CRD) of MR [26 ], the CRD that recognizes pathogen polysaccharides.

Although MR is known primarily for its function as a pattern recognition receptor (PRR), it also has an important role in tissue remodeling and lysosomal hydrolase and hormone clearance during inflammation and wound healing [27 28 29 ]. Although some studies indicate that redundancy in ligand specificity of PRR can counterbalance the lack of MR [12 , 13 ], it appears that the anti-inflammatory and scavenging properties cannot be compensated [13 , 28 ].

Despite the in vitro evidence suggesting a role for MR in phagocytosis of Leishmania parasites, the function of MR in host antileishmanial defense has never been determined in vivo. In light of this omission, coupled with evidence indicating an anti-inflammatory role for MR during wound healing, we dissected the role of MR during in vitro and in vivo immune responses against L. major and L. donovani infection using a MR-deficient murine model.

MATERIALS AND METHODS

Mouse strains
MR null (MR–/–) mice were generated by disrupting the gene Mrc1 on Chromosome 2, which encodes MR (CD206) by the insertion of a neomycin gene, and were the generous gift of Dr. Michel C. Nussenzweig (The Rockefeller University, New York, NY, USA). These mice were generated originally on a mixed strain of 129SvJ and C57BL/6 background and were backcrossed to the C57BL/6 strain for seven generations [28 ]. A MR wild-type (WT; +/+) line was generated from a MR hetrozygote (+/–) cross and maintained in the Freimann Life Science Center at the University of Notre Dame (Notre Dame, IN, USA) and the offspring genotyped by PCR. To detect the WT allele, the primer, MR-A 5'-GAC CTT GGA CTG AGC AAA GGG G-3', was used with MR-B 5'-GAC ATG ATG TCC TCA GGA GGA CG-3' to yield a band of 400 bp. Female MR-knockout (KO; –/–) and MR-WT (+/+) mice were used at 6–8 weeks of age. All animal protocols were reviewed and approved by the University of Notre Dame Institutional Animal Care and Use Committee. All experiments were in concordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Parasites and infections
L. major NIH Friedlin V1 strain (MHOM/IL/80/FN), isolated from a patient with localized CL in Israel and L. donovani 1S strain (MHOM/SD/62/1S) isolated from a VL patient in Sudan were used in this study. The Leishmania parasites were cultured at 26°C without CO2 in medium 199 (M199), supplemented with 20% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 40 mM HEPES, 0.1 mM adenine (in 50 mM HEPES), 5 mg/ml hemin (in 50% triethanolamine), and 1 mg/ml biotin. Infective-stage metacyclic promastigotes were isolated from stationary cultures (4–5 days old) by using a uniform procedure based on a modification of a method of density gradient purification described recently [30 ]. All parasites were tested for mycoplasma contamination [PCR detection method, TaKaRa (Bio Inc., Japan) and endotoxin (Limulus amoebocyte lysate assay, Endosafe Charles River Laboratories, Inc., Bar Harbor, ME, USA)].

For cutaneous infection of mice, L. major metacyclic promastigotes in 20 µl PBS were injected intradermally in the outside surface of the ear as described previously [31 ]. The following formula was used to estimate the volume of lesions: (Dmax/2xDmin/2xT/2) x 4/3 x {pi}, where Dmax = maximal diameter of the lesion; Dmin = minimal diameter of the lesion; and T = thickness of the lesion. The diameters and thickness of the lesion were measured using a dial gauge Vernier caliper. Visceral infections were initiated with L. donovani metacyclic promastigotes i.v. in the tail vein.

Estimation of parasite load
For parasite quantification in cutaneous lesions, each ear was peeled open, treated with 1 mg/mL collagenase (Gibco-BRL, Grand Island, NY, USA) for 2 h, and homogenized by cutting with sterile surgical scissors. For quantification of parasites in visceral organs, livers and spleens were harvested, treated with 1 mg/ml collagenase for 1.5 h, and homogenized with a syringe plunger, and RBC were lysed by associated tyrosine kinase lysis for 5 min. In each case, homogenized tissue was filtered through 70 µm cell strainers, and the filtrate was centrifuged to pellet parasites. These were resuspended in 200 µL M199 complete media and serially diluted 1:2 24 times in 96-well plates. Parasite burden was determined by calculating back from the last well that contained live parasites on Day 7.

Bone marrow-derived MP (BMDM) generation
Bone marrow cells were flushed from femurs and tibias of 6- to 8-week-old MR-WT and MR-KO by standard techniques [32 ]. The progenitor cells were incubated at 37°C with 5% CO2 in the presence of L929-conditioned medium (L-CM) in RPMI supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. L-CM, as a source of M-CSF, was added at a concentration of 30% and replaced every other day [33 ]. On the 7th day of culture, the adherent cells (BMDM) were harvested and infected; this population consists of mature MP, fibroblasts, and clusters of nonadherent cells. To assess phagocytosis (see Go Go Go Go Go Go Fig. 7 ), cells were cultured on coverslips. For some assays (see Fig. 1 ), the nonadherent cell clusters were harvested, disassociated, counted, and transferred to Chamber Slides (Lab-Tek, Naperville, IL, USA) on Days 5 and 6 of cultivation. These cells were then cultured for 2 additional days in the presence of 3% L-CM; these cells are denoted BMDMS, for BMDM, synchronized). As described previously, greater than 95% of these cells are mononuclear, nondividing, and have the phenotype of resident MP [34 ]. Flow cytometry analysis revealed that this population expresses MR to a similar level as BMDM described above (data not shown).


Figure 1
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  		Figure 1. MR-KO MP phagocytose Leishmania. MR-KO and MR-WT BMDMS were infected with L. major (A) or L. donovani (B) metacyclic promastigotes (7-1 parasites:BMDMS). Cells were washed at 0.5, 1, 2, and 4 h postinfection, and the number of parasites per 100 cells was assessed by light microscopy.

 

Figure 2
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  		Figure 2. Similar TNF-{alpha} and IL-12 production in MR-WT and MR-KO BMDM, which from MR-WT and MR-KO mice, were infected with L. major metacyclic promastigotes (10:1). Eighteen hours postinfection, cells were stimulated with 100 U/ml IFN-{gamma} for 4 h followed by 1 µg/ml bacterial LPS or mock-stimulated. Cells were cultured in 10 µg/ml Brefeldin A for the final 6 h. TNF-{alpha} (A) and IL-12 (B) production was assessed. The percentage of cytokine-positive F4/80 cells is indicated. Data presented are representative of three experiments. NON, Noninfected; INF, infected; S, stimulated; INF + S, infected and stimulated cells; SS, side scatter.

 

Figure 3
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  		Figure 3. MR is nonessential for Leishmania-induced inhibition of MAPK activation. BMDM from MR-KO and MR-WT mice were infected with L. major (Lm) and L. donovani (Ld) metacyclic promastigotes (20:1 parasite:MP ratio), stimulated with 1 µg/ml LPS for 1 h, or left untreated (U). MAPK activation was assessed by Western blot analysis using phospho-specific antibodies to ERK 1/2 (p-ERK1/2; A) and p38 (p-p38; B). Total levels of MAPK in each sample were also assessed. The data shown are representative of two independent experiments.

 

Figure 4
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  		Figure 4. MR-KO mice infected with L. major exhibit a similar clinical picture to control animals. MR-KO and MR-WT mice (10–15 mice per group) were infected intradermally with 2 x 105 L. major promastigotes. Increase in lesion volume was monitored by weekly measurement. The error bars represent the standard deviation of the mean. Data from one of three representative experiments are presented. *, P < 0.05.

 

Figure 5
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  		Figure 5. Inflammatory infiltrate from MR-KO and MR-WT L. major-infected mice exhibits similar characteristics. Mice were infected with 2 x 107 L. major per ear and 2 weeks later, assessed for leukocyte infiltration. (A) H&E staining of frozen ear skin section. Original magnification, x200. (B) MP (anti-F4/80-FITC; green) and DC (anti-CD11c-PE; red) in infected ear sections from MR-WT and MR-KO mice. (C) Number of neutrophils (anti-Ly-6G/Ly-6C) and MP (F4/80; green) in the dermis of the cutaneous lesions in MR-WT and MR-KO mice. (D) Flow cytometric analysis of MP (anti-F4/80 FITC) in cellular infiltrate from infected ears. Data from one of three representative experiments are presented.

 

Figure 6
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  		Figure 6. MR is dispensible for host defense against L. donovani infection. MR-KO and MR-WT mice (five to eight per group) were infected with 2 x 107 L. donovani metacyclic promastigotes. Liver (A) and splenic (B) parasite burdens were assessed by serial dilution assay 1, 2, 4, 8, and 12 weeks postinfection. Each circle represents an individual mouse, and bars indicate the mean value. •, MR-WT; {circ}, MR-KO. There was no significant statistical difference between MR-WT and MR-KO mice at any time-point. Data are presented from one of three independent experiments.

 

Figure 7
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  		Figure 7. Soluble mannan inhibits early uptake of Leishmania parasites. MR-KO and MR-WT BMDM were pretreated with 10 µg/ml soluble mannan prior to infection with L. major or L. donovani metacyclic promastigotes (10:1 parasites:BMDM). Cells were washed at 1 and 4 h postinfection, and the number of parasites per 100 cells was assessed by light microscopy. *, P < 0.05.

 
Western blot analysis
BMDM were infected with L. major or L. donovani metacyclic promastigotes at a 20:1 ratio (parasites:MP) for 1 h. Cells were washed with PBS to remove unattached parasites and lysed for 10 min in ice-cold lysis buffer (150 mM NaCl, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM pervanadate, 1 mM EDTA, 1% Igepal, 0.24% deoxycholic acid, 1 mM NaF, 50 mM Tris-HCl, pH 7.4). Lysates were stored at –20°C until use. Cell equivalents (5x104) in Lamelli buffer were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane, blocked with PBS, supplemented with 0.05% Tween 20 and 5% nonfat dried milk (PBST). Membranes were probed with primary antibodies against phospho-ERK1/2, total ERK1/2, phospho-p38, or total p38, purchased from Cell Signaling Technology (Beverly, MA, USA), or anti-GAPDH, purchased from Biogenesis (Kingston, NH, USA). Blots were washed in PBST and incubated with HRP-conjugated antirabbit or antimouse Ig (BD Biosciences, San Jose, CA, USA). Bound antibodies were detected with SuperSignal Pico or West Femto chemiluminescent substrate (Pierce, Rockford, IL, USA).

Immunohistochemistry and confocal microscopy
Cryostat cut sections (5 µm) of mouse ear were mounted in Tissue-Tec OCT (Miles, Elkhart, IN, USA) and were placed onto slides (Superfrost Plus microscope slides, Fisher Scientific, Pittsburgh, PA, USA) and allowed to dry for 15 min. Prior to immunostaining, the slides were placed into ice-cold acetone for 10 min following rehydration in PBS for 15 min. For immunohistochemistry, the sections were incubated with rat anti-F4/80 FITC (Caltag Laboratories, S. San Francisco, CA, USA) or anti-Ly6G/Ly-6C FITC or CD11c PE (BD Biosciences). After 1 h incubation (at 37°C temperature in a humidified chamber), the slides were washed in PBS for 10 min and fixed with Fluoromount-G (Southern Biotechnology Associates, Inc., Birmingham, AL, USA). The samples were analyzed with a Nikon Diaphot 200 confocal laser fluorescence microscope. For characterization of cell components of the inflammatory infiltrate, cell counting was performed. Only cells with a visible nucleolus and showing clear immunostaining were counted as positive. All fields of interest were counted at a magnification of 600x, and the amount of positive cells was presented as number of positive cells/mm2.

Analysis of intradermal leukocytes
To characterize leukocytes in the inoculation sites, ears were collected, and the ventral and dorsal dermal sheets were separated and incubated dermal-side-down in RPMI 1640, NaHCO3, and penicillin/streptomycin/gentamicin, containing 125 U/ml collagenase A (Sigma-Aldrich, St. Louis, MO, USA) for 2 h. The dermal sheets from five to seven animals were pooled, cut into small pieces, and filtered through a 70-µm nylon cell strainer before being washed twice in RPMI 1640, NaHCO3, penicillin/streptomycin/gentamicin, 10% FCS, and 0.05% DNase I (Sigma-Aldrich).

Phagocytosis assay
BMDMS (2x105) were cultured in eight-well chamber slides, or BMDM were generated on coverslips. An infection ratio of 5-7 parasites per one MP was used. For some studies, cells were opsonized with 5% normal mouse serum prior to infection. The percentage of infected cells and the number of parasites per infected cell were determined by Diff-Quik staining and light microscopy. Intracellular parasites were distinguished from extracellular parasites by a lack of a flagellum and a vacuole surrounding the parasite. Infection rates were determined at 0, 0.5, 1, 2, and 4 h. For blocking studies with soluble mannan, cells were pretreated with 10–1000 µg/ml soluble mannan.

Flow cytometry
Cells were analyzed by flow cytometry for surface markers and cytoplasmic staining for cytokines. The following antibodies were used: anti-TNF-{alpha}-PE (MP6-XT22) and anti-IL-12p40/p70-PE (C15.6) from BioLegend (San Diego, CA, USA) and anti-F4/80 FITC from Caltag Laboratories. For intracellular cytokine production, BMDM were generated as above and infected at a 10:1 ratio for 4 h, subsequently stimulated with 100 U/ml IFN-{gamma} overnight and then 1 µg/ml bacterial LPS (Sigma-Aldrich) for 8 h. Brefeldin A (10 µg/ml) was added the final 6 h of culture. Finally, cells were harvested 24 h after the infection was initiated. Cells were washed, fixed in 1% paraformaldehyde, permeablized with 0.1% saponin, and stained with antibodies conjugated directly to fluorescent dye. A similar protocol was used for cell surface staining of ear leukocytes but without Brefeldin A and saponin.

Statistical analyses
A one-way ANOVA and a subsequent Tukey’s Honestly Significantly Different test was used on Log10 transformed data for statistical analyses of the in vivo experiments, and a Student’s t test was used for in vitro experiments using GraphPad Prism Version 4 software. In all cases, a Pvalue of less than 0.05 was considered statistically significant.

RESULTS

MR-deficient MP exhibit a similar infection rate in comparison with MR-WT BMDMS
Previous studies using MR ligands to block Leishmania attachment have implicated MR in internalization of Leishmania promastigotes [6 , 15 , 16 , 22 ]. To determine if MR plays a significant role in the uptake of L. major and L. donovani, in vitro phagocytosis assays were performed. BMDMS from MR-WT and MR-KO animals were generated and infected with unopsonized L. major and L. donovani parasites. No significant differences were observed 4 h after L. major infection (118.19±10.18 in MR-WT and 152.34±31.52 in MR-KO; Fig. 1A ) or L. donovani infection (126.19±29.88 in MR-WT and 123.80±3.00; Fig. 1B ) in both strains of mice. Opsonization with normal mouse serum increased phagocytosis dramatically (two- to tenfold), an increase that was not affected by the absence of MR (data not shown). Furthermore, the kinetics of infection did not vary in the absence of MR, indicating that MR is not necessary for internalization of Leishmania in MP.

MR deficiency does not influence the production of TNF-{alpha} and IL-12
Treatment of MP with soluble mannan leads to TNF-{alpha} production [35 ], suggesting that interaction with MR at the cell surface may be responsible for TNF-{alpha} production in response to Leishmania infection. To determine the role of MR in cytokine production during Leishmania infection, we assessed TNF-{alpha} (Fig. 2A ) and IL-12 (Fig. 2B) in MR-WT and MR-KO BMDM infected with L. major by flow cytometry. Intracellular cytokine staining revealed that BMDM from MR-WT and MR-KO mice produce similar amounts of TNF-{alpha} and IL-12 during L. major infection, indicating that MR engagement by L. major is not required for production of these cytokines. Leishmania infection renders MP unable to produce high amounts of IL-12 in response to proinflammatory stimuli including bacterial LPS, Staphylococcus aureus, mycobacteria, IFN-{gamma}, and CD40 ligand [36 37 38 39 40 ]. This inhibitory phenotype has been attributed to parasite binding to specific host cell receptors, which send a negative signal. MR, specifically, has been implicated in blocking LPS signals and IL-12 production in dendritic cells (DC) [41 , 42 ] but has not been assessed in MP. Our experiments revealed that a MR interaction is not essential for the inhibition of LPS-induced IL-12 production by L. major infection in BMDM (Fig. 2B) ; the percentage of LPS-induced, IL-12-producing cells was decreased by 41.0% for MR-WT and 54.0% for MR-KO in Leishmania-infected cells as compared with noninfected cells.

Leishmania parasites evade activation of MAPKs in MR-WT and MR-KO BMDM
L. donovani parasites have been reported to enter naïve MP silently, avoiding the activation of the ERK1/2, p38, and JNK kinases; this avoidance of MAPK activation has been attributed to the presence of LPG on the parasite surface [43 ]. Here, we show that L. major and L. donovani avoid activating ERK1/2 (Fig. 3A ) and p38 (Fig. 3B) during infection. We demonstrate further that interaction with MR is not necessary for inhibition of ERK and p38 activation.

Normal healing process and clearance of L. major in MR-deficient mice
To assess the tissue-remodeling properties of MR during Leishmania infection, the clinical course of L. major infection in MR-WT and MR-KO mice was investigated. Inoculation of 2 x 105 promastigotes into the ear dermis led to slow lesion development that peaked at ~4 weeks and resolved slowly over the subsequent 8 weeks in MR-WT and MR-KO mice (Fig. 4 ). There was a slight difference in the volume of lesions between MR-WT and MR-KO mice at Week 7; however, the terms of ulcer healing and resolution of lesions were equivalent. Reinfection after healing revealed dramatically smaller lesions and increased healing kinetics, and no difference was between MR-WT and MR-KO mice in terms of a clinical picture of CL (data not shown). Parasite quantification from infected ears was performed during healing of ulcerative lesions at Week 9. No significant differences were observed between parasite replication in the ears of MR-deficient and control animals (data not shown).

Characteristics of inflammatory infiltrate and number of MP in the inflammatory infiltrates from MR-WT and MR-KO are comparable
A well-formed inflammatory infiltrate in the deep layers of the dermis with formation of isolated granulomas was observed in both strains of mice (Fig. 5A ). The infiltrate was characterized by a predominance of mononuclear cells, and there was no difference in the architecture of lesions from MR-WT and MR-KO mice. Immunotyping analysis revealed that neutrophils were the major type of cells in the inflammatory infiltrate, followed by MP (neutrophils as Ly-6G/Ly-6C-positive cells were 1114±82 cells/mm2 in MR-WT and 1057±94 cells/mm2 in MR-KO; MP as F4/80-positive dermal cells were 845±92 cells/mm2 in MR-WT and 801±146 cells/mm2 in MR-KO; Fig. 5C ).

As MP play the major role in the eradication of the Leishmania infection [44 ], we quantified the number of MP (F4/80-positive cells) in the inflammatory infiltrate induced by L. major infection by flow cytometry. Equal amounts of MP were detected in the inflammatory infiltrate from MR-WT and MR-KO animals (Fig. 5D) . Confocal microscopy was used to identify the distribution of MP (F4/80-positive) and DC (CD11c-positive) within the lesion of infected mice (Fig. 5B) . We did not observe any unequal accumulation of MP or DC in MR-WT and MR-KO mice.

Normal parasite clearance during experimental VL in MR-deficient mice
To assess the role that MR plays during VL infection, MR-WT and MR-KO mice were i.v.-infected with 2 x 107 L. donovani metacyclic promastigotes, and parasites were quantified in visceral organs over 12 weeks of infection (Fig. 6 ). Although parasite levels in MR-KO mice appear lower at some time-points, the trend was not maintained in all experiments and was never statistically different than the levels detected in MR-WT mice.

Soluble mannan partially inhibits Leishmania uptake in the absence of MR
As previous studies have blocked Leishmania entry with soluble mannan, we assessed L. major and L. donovani metacyclic promastigote entry into WT- and MR-deficient BMMP (Fig. 7 ). We observed a partial inhibition of parasite uptake by mannan (10 µg/ml) pretreatment early during in vitro infection of both strains of mice, indicating that mannan is blocking parasite uptake via a receptor other than MR. Similar results were observed when 1 mg/ml mannan was used to block parasite entry (data not shown).

DISCUSSION

Terminal mannosylation of polysaccharides is a common motif among pathogenic organisms, including protozoa, bacteria, fungi, and viruses. Recognition of such surface patterns that are common among pathogens is how the innate immune system is able to identify potential threats. MR is one such PRR that recognizes repeated mannose-containing moieties. The theory that MR is involved during Leishmania infection is supported by indirect evidence involving blocking parasite attachment in vitro with MR ligands, specifically, soluble mannan [6 , 15 , 16 , 22 ]. The present work allowed the direct assessment of MR in Leishmania entry, cytokine production, and disease resolution.

Our results show that MR-deficient BMDM phagocytose L. major and L. donovani metacyclic promastigotes efficiently, suggesting that redundant MP receptors compensate for the lack of MR or that MR plays no role in parasite attachment. We demonstrate further that MR is dispensable for proinflammatory TNF-{alpha} production in response to Leishmania infection. At first glance, these results appear in opposition to the literature published previously, suggesting MR involvement [6 , 15 , 16 , 22 ]. However, recent evidence indicates that other C-type lectin receptors recognize mannose-bearing ligands [45 ]. Furthermore, TLR family members are able to bind mannan [46 ]. TLR2 has been implicated specifically in MP responses to L. major [47 ] and L. donovani [48 ]. Likely, use of mannan to block pathogen internalization interferes with multiple ligand-receptor interactions, as our results demonstrate that soluble mannan blocks parasite uptake even in the absence of MR.

Leishmania promastigotes enter host MP in a relatively silent manner, avoiding the signaling cascades associated with activation [43 ]. This inhibition requires the presence of virulence glycoconjugates on the parasite surface; the most abundant factor exhibiting these specialized moieties is LPG, which forms a dense glycocalyx that covers the promasitgote surface, making LPG the major factor interacting with receptors on the surface of the host cell. The presence of mannose in the repeating units of LPG and other Leishmania glyconjugates suggests that these factors may be recognized by MR. Our studies assessing ERK1/2 and p38 phosphorylation in MR-KO MP indicate that an interaction between Leishmania and MR is not necessary for the inhibition of MAPK activity. MAPK activation by L. donovani in the absence of LPG occurs within 5 min [43 ], indicating that evasion of MAPK activation is likely initiated at the cell surface. The data presented here suggest that MR plays no role in the inhibitory process, or redundant receptors compensate for the lack of MR in our system.

The expression of MR is dynamic during immune responses [49 50 51 ]. MR is expressed highly during the initial stages of immune reactions to enable MP to internalize antigens for presentation to T lymphocytes. Subsequent IFN-{gamma} production from T cells down-regulates MR and results in the release of reactive oxygen intermediates, lytic enzymes, NO, and cation proteins to the intercellular space. These reactive species cause pathology to the surrounding tissues. Prostaglandins, IL-4, and IL-13 are associated with the negative-feedback loop that is initiated for the resolution of inflammation after infection. These mediators restrict self-damage by down-regulating the expression of NO, promote repair, and up-regulate MR expression. MR is an important receptor for clearance of endogenous inflammatory enzymes (lysosomal hydrolases, myeloperoxidase) and a tissue plasminogen activator [27 , 52 53 54 ] and thus initiates the final resolution of immune responses and wound healing. This role suggests that MR-KO mice should exhibit a delay in healing of Leishmania lesions. However, our data reveal that a deficiency of MR does not alter susceptibility to infection with L. major in the MR-KO mouse. The clinical dynamics and tissue-parasite burdens after healing of cutaneous lesions for MR-WT and MR-KO failed to reveal a consistent pattern of significant differences, indicating an unaltered level of host resistance against the pathogen in the absence of MR expression. Furthermore, our data indicate that MR is nonessential for resistance to VL.

Cell-mediated immunity is generally believed to be of a dominant influence in determining the outcome of leishmanial disease. Th1 responses protect against infection with L. major, whereas susceptibility is related to a Th2 response [55 ]. In this context, it has been found that L. major metacyclic promastigotes actively suppress production of the proinflammatory cytokine IL-12 in BMDM [38 , 56 ]. As IL-12 is a major, physiological promoter of IFN-{gamma} production, and Leishmania is highly susceptible to killing by IFN-{gamma}-activated MP, this ability to suppress IL-12 production would be expected to provide a clear survival advantage to the parasite. Uptake of parasites through specific PRR can be one of the mechanisms of IL-12 suppression, and MR has been implicated in this process [41 ]. It has been shown that mannan (which is a natural ligand for MR) and mAb specific for MR inhibit IL-12 production in human APC [41 ]. In our experiments, we observed a similar level of IL-12 inhibition in MR-WT and MR-KO mice. The numbers of IL-12-positive BMDM after L. major infection and stimulation by IFN-{gamma} and LPS were reduced and comparable between MR-KO and MR-WT mice. These data indicate the redundant mechanisms of IL-12 regulation and suggest the importance of other factors and receptors (CR3 and FcR) in the parasite-dependent blockage of IL-12 production [57 , 58 ].

In summary, our data suggest that MR is not essential for host resistance against Leishmania infections and suggest that although MR may influence host defense subtly, engagement of other PRR, previously shown to engage Leishmania parasites, such as CR3 [8 ], FcR [9 ], and TLR [47 , 48 ], is likely responsible for cytokine production and inhibition of MP responses.

ACKNOWLEDGEMENTS

The University of Notre Dame, College of Science, provided funding. The University of Notre Dame Graduate School supported O. E. A. We are grateful to Dr. Michel C. Nussenzweig (The Rockefeller University) for the use of MR-KO mice. We thank Freimann Life Science Centre, particularly Lindsay N. Barnett and June Peer, for excellent animal care, Michael J. Donovan for help with bone marrow assays, and James P. Whitcomb for mouse genotyping. We are also grateful to Dr. Kevin Drury for his advice on the statistical analysis.

FOOTNOTES

1 These authors contributed equally to this work. Back

2 Current address: Wellman Center for Photomedicine, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, 40 Blossom St., Bartlett 404C, Boston, MA 02114, USA. Back

Received July 10, 2006; revised December 28, 2006; accepted December 29, 2006.

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