7–10 mL of peripheral blood samples were collected from patients suffering from CL and Ethiopian healthy controls (EHCs) living in Oromo and Amhara regions of Ethiopia. The sample collection was permitted based on the local ethical committee (allowance number PO25/08, Addis Ababa, Ethiopia) and the national health research ethics review committee (approval number 310/227/07, Addis Ababa). Written informed consent was obtained from the participants of this study. CL was confirmed by positive parasite cultures and PCR analysis as described elsewhere (49). CL patients were excluded from the study if they show one of the following criteria: younger than 18 and older than 55 years, chronic lesions (more then 6 months), positive for HIV, clinical evidence for coinfections, and drug intake.

Female wild-type BALB/c and C57BL/6 mice (Janvier Labs, Le Genest St. Isle, France) were kept at the animal facility of the University of Regensburg under pathogen-free conditions. Dectin-1^−/− mice on BALB/c background [kindly provided by G.D. Brown, University of Aberdeen (50)] were bred and maintained under conventional animal housing conditions. All experiments and animal housing were performed according to the guidelines for the care and use of experimental animals. The animal work was approved by the local veterinary authorities of the district government based on the European guidelines and national regulations of the German Animal Protection Act (approval no. AZ 54-2532.105/11). Female animals between 6 and 12 weeks of age were used for experiments.

Virulent L. major parasites (MHOM/IL/81/FE/BNI) were propagated in vitro in blood agar cultures as described previously (51). Stationary phase promastigotes from the third to seventh in vitro passage were harvested, washed four times, and resuspended in PBS. Mice were infected via intradermal injection of 3 × 10⁶ stationary phase promastigotes in 30 µL into the hind footpads. The increase in lesion size was monitored weekly by measuring the footpad thickness with a metric caliper (Kroeplin Schnelltaster, Schlüchtern, Germany). The increase in footpad thickness (%) was determined as described elsewhere (52).

Curdlan (WAKO Chemicals GmbH, Neuss, Germany) was dissolved in sterile PBS to a concentration of 50 µg/µL. 3 × 10⁶ stationary phase promastigotes were resuspended in 30 µL of Curdlan solution and injected intradermally into the hind footpads. Alternatively, soluble L. major antigens [SLA; (15)] corresponding to 3 × 10⁶ stationary phase promastigotes were used. The course of disease was monitored as described earlier.

Serum from naïve mice and from mice infected with L. major parasites was prepared at the corresponding time point and thereafter stored at −20°C until use. Detection of L. major-specific IgG₁ (Invitrogen, Darmstadt, Germany), IgG_2a (BD Pharmingen, Heidelberg), and IgG_2c (Jackson ImmunoResearch, Hamburg Germany) isotypes were performed as described earlier (53). The results were standardized by calculation of relative ELISA units (REU). REU were determined by the formula: OD₄₅₀ serum (infected mice)/OD₄₅₀ serum (naïve mice).

Three weeks after L. major infection, SLA (corresponding to 3 × 10⁶ parasites) was injected in a volume of 20 µL in the foreleg. As a control, 20 µL of PBS was injected in the contra lateral foreleg. The increase in swelling was measured with a metric caliper (Kroeplin Schnelltaster) and referred to the control foreleg. The swelling was measured with a metric caliper (Kroeplin Schnelltaster) after 24, 48, and 72 h.

Bone marrow-derived dendritic cells were generated as previously described (54). Bone marrow cells from C57BL/6 or BALB/c mice were seeded in 10 cm BD Tissue culture dishes at a density of 2 × 10⁶ per dish in 10 mL of RPMI medium supplemented with 10% fetal calf serum (FCS; PAN Biotech GmbH), penicillin–streptomycin, 50 µM β-ME, and 10% GM-CSF containing supernatant harvested from Ag8653 myeloma cells transfected with the gene encoding murine GM-CSF (kindly provided by B. Stockinger, NMRI, Mill Hill, London, UK). BMDCs were harvested on day 10 for T cell proliferation experiments.

Mouse BMDMs were generated from female BALB/c mice and cultured for 7 days in hydrophobic Teflon bags (FT FEP 100C Dupont, American Durafilm, Holliston, MA) as described earlier (55, 56). For killing experiments, BMDMs were pulse-infected with L. major promastigotes at a 1:30 ratio for 4 h as described earlier (56). After infection, extracellular promastigotes were removed by washing with PBS and cultured in the absence or presence of Curdlan (50, 100, and 250 µg/mL) or LPS/IFN-γ (10 and 20 ng/mL, respectively). After 72 h, BMDMs were fixed, stained, and analyzed microscopically using Diff-Quick staining (Medion Diagnostics, Düdingen, Switzerland) for the determination of the percentage of infected cells and the number of parasites per infected cell. LPS (Escherichia coli O111:B4) was purchased from Sigma-Aldrich (Taufkirchen, Germany). Recombinant murine IFN-γ was purchased from eBioscience (Frankfurt, Germany).

Nitrite accumulation in the supernatants was determined as an indicator for NO activity after stimulation of BMDMs using the Griess reaction as described earlier (56).

Stationary phase promastigotes from the third to seventh in vitro passage were harvested, washed four times, and resuspended in PBS. After lysis of the remaining erythrocytes, parasites were labeled by incubation in 1 µM CFSE staining solution as described earlier (57).

C57BL/6 and BALB/c mice were infected with L. major parasites as described earlier. Ten days post infection, SDLNs were removed, and T cells were enriched untouched via MACS separation columns (Miltenyi Biotec, Bergisch Gladbach) according to the manufacturer’s guidelines. Single-cell suspensions were labeled with PE-conjugated CD11b, CD11c, and B220 antibodies (Abs) and subsequently detected with anti-PE Beads (Miltenyi Biotec) in order to deplete myeloid cells and B cells. The purified T cells were labeled with CFSE as described earlier (15). 2 × 10⁵ CFSE-labeled T cells were seeded in 96-well round-bottom plates (Nunc) to 2 × 10⁴ BMDCs that have been primed for 24 h with SLA (as an equivalent of 5 parasites: 1 BMDC) or L. major parasites (10 parasites: 1 BMDC) in the presence or absence of Curdlan (50 μg/200 μL). 72 h after coculture, the cells were analyzed by flow cytometry. The proliferation index of samples was calculated according to the formula: (number of proliferating cells after stimulation)/(number of proliferating cells in the absence of stimulation). The proliferation index of stimulated samples was then normalized to that of unstimulated BMDC/T-cell cultures. Supernatants were collected and stored at −80°C for cytokine analysis.

The CLR-Fc fusion proteins (Dectin-1, CLEC-9a, and MGL-1) were prepared as described previously (58). After blocking (PBS/10% FCS/10% mouse serum), L. major parasites were washed three times in PBS. To analyze interactions with CLRs, parasites were incubated with 20 µg/mL of CLR-Fc fusion proteins diluted in lectin binding buffer (50 mM HEPES, 5 mM MgCl₂, and 5 mM CaCl₂, in dH₂O, pH 7,4) at 4°C for 1 h. After three washing steps with PBS, CLR-Fc binding to L. major was detected with a FITC-conjugated goat anti-hFc Ab (Dianova, Hamburg, Germany). Parasite DNA was detected with 6-diamidino-2-phenylindole obtained from Sigma Aldrich, as described earlier (59). After mounting with PermaFluor (Thermo Scientific, Dreieich, Germany), the sections were analyzed using Axio Imager.M1 (Zeiss, Jena, Germany) equipped with high-sensitivity gray scale digital camera (AxioCam MRm, Zeiss). Separate images were collected for each section, analyzed, and merged afterward (acquisition software: Zeiss AxioVision 4.6.3). Final image processing for illustrations was performed using Adobe Photoshop Elements (Adobe Systems GmbH, Munich, Germany).

Single-cell suspensions of tissues were generated as described elsewhere (60). The following Abs and reagents were used for phenotyping of single-cells: PE-labeled anti-mouse CD11c Ab (clone N418; eBioscience, Frankfurt, Germany), APC-labeled anti-mouse CD11b (clone M1/70, eBioscience, Germany), PE-labeled anti-mouse F4/80 (clone 521204, R&D), biotinylated anti-mouse Dectin-1 Ab (clone 2A11; Bio-Rad AbD Serotec GmbH, Puchheim, Germany), AlexaFluor647-labeled anti-mouse CD86 Ab (clone GL-1; BioLegend, San Diego, CA, USA), and FITC-labeled anti-BrdU Ab (clone B44; BD Pharmingen, Heidelberg, Germany). Depending on the experimental setup, the detection of biotinylated anti-mouse Dectin-1 was performed using streptavidin-conjugated to V500 (BD, Pharmingen, #561419), eFluor450 (eBioscience, #48-4317-82), Pacific Orange (Thermo Fisher, Munich, Germany), or PerCP (BD, Pharmingen, #554064). Ab specificity was verified using appropriate isotype controls and Dectin-1^−/− mice (Figure S1 in Supplementary Material). Multicolor flow cytometry was performed as described previously (13). Mice were fed with BrdU-containing drinking water (0.8 mg/mL, Sigma, Deisenhofen, Germany) starting 3 days before the termination of the experiment. BrdU labeling was performed according to the manufacturer’s instructions (BD Pharmingen, Heidelberg, Germany). After labeling of surface epitopes, the cells were fixed and permeabilized with Cytofix/Cytoperm buffer (BD Pharmingen, Heidelberg, Germany) for 30 min on ice and afterward incubated for 10 min on ice with 0.01% Triton X-100 (SERVA, Heidelberg, Germany) in PBS/0.1% BSA (PAN, Aidenbach, Germany). For the detection of incorporated BrdU, the cells were treated with 30 µg DNAse (Sigma, Munich, Germany) for 1 h at 37°C. Labeling of cells with anti-BrdU Ab was performed for 20 min at room temperature. Cells were collected using BD™ LSRII Flow Cytometer (BD Biosciences, Heidelberg, Germany) and analyzed with FlowJo software (Tree Star Inc., Ashland, OR, USA). The determination of the total cell numbers in tissues was performed as described earlier (61).

Surface antigens were detected on fresh cells using a modified version of the method described by Aldebert et al. (62). 7–10 mL of blood from patients and EHCs were taken with BD Vacutainer (BD Biosciences, UK) containing 170 IU of lithium heparin. 200 µL of whole blood cells was incubated for 20 min with FcR blocking reagent (Miltenyi Biotec GmbH, Germany). The staining was performed for 30 min at 4°C. The following Abs were used: FITC-labeled anti-lineage (Lin) panel (CD3, CD14, CD16, CD19, CD20, and CD56) (BD), PerCP-labeled anti-HLA-DR (R&D, clone L203), PE-labeled anti-CD123 (R&D, clone 32703), PE-Cy5.5-labeled anti-CD11c (BD, clone B-Ly6), and APC-labeled anti-Dectin-1 (R&D, clone 259931). Erythrocytes were lysed with BD FACS™ Lysing Solution (BD Biosciences), and the remaining white blood cells were washed and resuspended in FACS buffer (PBS buffer, pH 7.2 1% BSA fraction V, Roth, 8076.2), 0.01% NaN₃ (Sigma: S2002-25G), and 1% human serum. A total of 200,000 events were acquired and the positive population gated using the isotype controls for the different fluorochromes. Data were acquired with a FACS Canto flow cytometer (BD Bioscience). Data analysis was performed with FlowJo 8.8.6.

CD4⁺ T cells were sorted with the CD4⁺ Cell isolation Kit^® (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s specification. Following cell sorting, the CD4⁺ cells were lysed in 350 µL Buffer RA1 (Macherey-Nagel, Düren, Germany) supplemented with 3.5 µL β-ME. Total RNAs were isolated using the NucleoSpin^® RNA kit (Macherey-Nagel), following the manufacturer’s instructions. RNAs were quantified by spectrophotometry and 170 ng of RNAs were used for cDNA synthesis using the iScript cDNA Synthesis kit (Bio-Rad Laboratories, München, Germany), as recommended by the manufacturer. Quantitative PCR was performed on a RotorGene Q (Qiagen, Hilden, Germany) using a two-step PCR program (95°C 15 s, 60°C 60 s; 40 cycles). 20 µL quantitative PCR reactions were performed using 0.3–0.6 µL of cDNA template (corresponding to 2.5–5 ng RNA equivalent) and a self-made master-mix containing SYBR Green I and HotStarTaq DNA Polymerase (Qiagen). Data were normalized to GAPDH mRNAs and expressed as relative mRNA levels, as previously reported (63, 64). Forward (fwd) and reverse (rev) mouse-specific quantitative PCR primers were as follows: GAPDH, 5′-AGCTTGTCATCAACGGGAAG-3′ (fwd) and 5′-TTTGATGTTAGTGGGGTCTCG-3′ (rev); GATA, 5′-CCAAGGCACGATCCAGCACAGA-3′ (fwd) and 5′-GGCCGACAGCCTTCGCTTGG-3′ (rev); IFNG, 5′-AGGTCAACAACCCACAGGTCC-3′ (fwd) and 5′-GATTCCGGCAACAGCTGGT-3′ (rev); IL-4, 5′-ACAGGAGAAGGGACGCCAT-3′ (fwd) and 5′-GAAGCCCTACAGACGAGCTCA-3′ (rev); IL-17A, 5′-AACTCCCTTGGCGCAAAAG-3′ (fwd) and 5′-GAGAGTCCAGGGTGACGTGG-3′ (rev); STAT4, 5′-CCTGGGTGGACCAATCTGAA-3′ (fwd) and 5′-CTCGCAGGATGTCAGCGAA-3′ (rev); STAT6, 5′-CCCCAACAAACTTCTCATCCA-3′ (fwd) and 5′-TTTGGCGTTGTTGTCTTGGTT-3′ (rev); and T-BET, 5′-ACCAACAACAAGGGGGCTCT-3′ (fwd) and 5′-CTCTGGCTCTCCATCATTCACC-3′ (rev).

To measure the parasite burden, genomic DNA was isolated using DNA purification solutions from QIAGEN (QIAGEN, Hilden, Germany). In brief, the cells were digested in cell lysis solution directly in the well. Protein was removed by adding protein precipitation buffer, and DNA was precipitated according to the manufacturer’s instructions with 100% 2-propanol (Sigma Aldrich, Taufkirchen, Germany). The concentration of mouse β-actin-DNA was quantified by PCR (65). The Leishmania DNA concentrations in the same samples were determined using fluorescence resonance energy transfer real-time PCR with Leishmanial 18S ribosomal DNA sequences (66). The resulting Leishmania DNA copy number was then divided by the copy number of β-actin-DNA to obtain the relative parasite units (RPU).

Supernatants of cell cultures were analyzed with a FlowCytomix™ kit (IL-13, IL-1α, IL-22, IL-2, IL-21, IL-6, IL-10, IL-27, IFN-γ, TNF-α, IL-4, and IL-17) according to the manufacturer’s (eBioscience, Thermo Fisher Scientific, Waltham, USA) specifications. Acquisition of probes was performed with a FACS Canto II (BD Bioscience).

Statistical significance between groups were calculated by different tests: two-way ANOVA for comparing the footpad swelling of analyzed groups, non-parametric Mann–Whitney tests if no normal distribution (no Gaussian distribution) was determined, and two-tailed Student’s t-test if normal distribution (Gaussian distribution) was determined. The calculation was performed using PRISM software (GraphPad, La Jolla, CA, USA). The corresponding p values are highlighted by stars only if p < 0.050.

DCs are known to be important checkpoints for the generation of T cell-mediated protective immunity in experimental leishmaniasis (67). Based on the potential role of Dectin-1⁺ DCs in adaptive immunity, we addressed the question whether a cutaneous infection with Leishmania parasites results in an expansion of Dectin-1⁺ DCs.

In humans, it is difficult to analyze the dynamics of DC populations in tissues or secondary lymphoid organs. Therefore, we focused on the analyses of blood samples. DCs in the peripheral blood lack certain Lin-specific markers (68). Thus, Lin⁻ leukocytes were used for further characterization of human DCs (Figures 1A,B). The Lin⁻ population consists of HLA-DR⁻ and HAL-DR⁺ cells (Figure 1B). In accordance with other published data, the Lin⁻ CD11c⁺ cells are positive for HLA-DR and could be found within the Lin⁻/HLA-DR⁺ population (68, 69) (data not shown).

On the basis of the markers CD11c and CD123 (68, 70) myeloid DCs (mDC; CD11c⁺/CD123^−/int) were dissected from plasmacytoid DC (pDC; CD11c⁻/CD123^bright) subsets (Figure 1C). We could demonstrate that pDCs from EHCs and CL patients hardly express Dectin-1 (Figure 1D). The expression of Dectin-1 is predominantly restricted to mDCs (Figure 1D). Cutaneous infection with Leishmania parasites results in an increased frequency of Dectin-1⁺-positive mDCs in the periphery compared to healthy controls (Figure 1E). Of note, the pDC subset does not show significant differences in Dectin-1 expression between EHCs and patients suffering from CL (Figure 1F). These data suggest that a local infection with Leishmania parasite results in the expansion of Dectin-1⁺ mDC subsets in the peripheral blood of CL patients.

CD11c is hardly expressed by pDCs in mouse models (71, 72). Thus, most of the CD11c^+/bright DCs belong to the subset of conventional DCs including other mDC subsets (73, 74). In CL patients, Dectin-1⁺ mDCs expand after infection. To prove the concept that a local infection also results in a systemic expansion of Dectin-1⁺ mDCs, C57BL/6 and BALB/c mice were infected intradermally with L. major parasites. SDLNs, the site of infection, and the peripheral blood were analyzed for proliferating Dectin-1⁺ CD11c^+/bright DCs. Mice were administered the thymidine analog BrdU, as described in Section “Materials and Methods,” to analyze cell proliferation in vivo. 13 days after infection, when clinical symptoms such as footpad swelling occur and Leishmania-specific T cell-mediated immunity is initiated (13, 60), Dectin-1 was measured on BrdU⁺ (proliferating) and BrdU⁻ (resting) CD11c⁺ DCs (Figures 2A,B). Detailed quantification of Dectin-1⁺ DCs within proliferating and resting CD11c⁺ DCs revealed that both subsets express Dectin-1. However, the frequency of Dectin-1⁺ CD11c⁺ DCs is substantially higher within the proliferating subset compared to the resting CD11c⁺ DCs of BALB/c and C57BL/6 mice (Figures 2C,D). Further analysis of the site of infection and SDLNs of infected BALB/c and C57BL/6 mice confirmed this result (Figures S2A,B in Supplementary Material). These data generated from mice reflect the results obtained in CL patients, indicating that a local infection with Leishmania parasites results in a systemic expansion of Dectin-1⁺ DCs.

So far, only systemically (i.p. and/or i.v.) applied β-glucans have been used to treat BALB/c or C57BL/6 mice after infection (34, 43–48). No published data exist analyzing the effects of cutaneous application of β-glucan such as Curdlan in parallel to L. major infection. Given that Dectin-1⁺ DCs expand at the site of infection and within SDLNs (Figure 2; Figure S2 in Supplementary Material), it seems feasible that a local stimulation of Dectin-1⁺ DCs with Curdlan modulates the parasite-specific immune response. To test this hypothesis, BALB/c and C57BL/6 mice were infected with promastigote parasites suspended in PBS plus Curdlan. The course of infection was not substantially modulated in resistant C57BL/6 mice (Figure S3 in Supplementary Material). In contrast, normally susceptible BALB/c mice showed a resistant phenotype when the parasites were intradermally injected in combination with Curdlan (Figure 3A). BALB/c mice infected with L. major alone displayed the well-known severe course of disease (Figure 3A). An additional control experiment with Dectin-1^−/− BALB/c was performed to confirm the specificity of the Curdlan/Dectin-1 interaction in vivo. Here, we were able to demonstrate that an application of Curdlan together with the parasites does not result in protective immunity against L. major parasites as shown in protected BALB/c wild-type mice (WT) (Figure 3A; Figure S4 in Supplementary Material). The experiments with non-healing BALB/c WT and Dectin-1^−/− mice had to be terminated based on the severe ulcerated footpads.

C57BL/6 mice usually resolve a primary infection with L. major. Following resolution of the primary infection, they are immune to reinfection due to the generation of antigen-specific memory T cells (75, 76). To test whether Curdlan-protected BALB/c mice (Figure 3A) show classical signs of adaptive immunity, reinfection experiments were performed. BALB/c mice that had received Curdlan intradermally in the absence of parasites were used to exclude the possibility of an unspecific expansion of T cells potentially mediating protection. Resistant BALB/c mice, that had been infected with L. major parasites in the presence of Curdlan, were reinfected into the contra lateral footpad at day 131 after primary infection with a high-dose of parasites in the absence of Curdlan. At this time point, no clinical signs such as footpad swelling were detectable. BALB/c mice that have been treated intradermally only with Curdlan 131 days before the primary infection developed a severe course of disease, that is, comparable to infected BALB/c mice that were not pretreated (Figure 3B). In contrast, protected BALB/c mice, that resolved the primary infection, were able to rapidly control the second round of a high-dose infection with Leishmania parasites in the absence of Curdlan (Figure 3B). These mice now show symptoms of an infection until the termination of the experiment 120 after the second exposition to Leishmania parasites in the absence of Curdlan.

Beside a reinfection, the DTH response is also an accepted indicator for the presence of an adaptive T cell-mediated immune response (75, 77). In contrast to resistant C57BL/6 mice, BALB/c mice hardly generate a measurable DTH reaction after a high-dose infection with L. major parasites (52). Using the high-dose model, we were able to demonstrate that Curdlan-protected BALB/c mice can indeed develop a DTH reaction that is comparable to that of resistant C57BL/6 mice (Figure 3C). Consequently, protected BALB/c mice mount an adaptive T cell-mediated immunity against L. major parasites that is accompanied by a clear reduction of the parasite load at the site of infection during the effector phase of experimental leishmaniasis (Figure S5 in Supplementary Material).

It needs to be mentioned that BALB/c mice can develop protective immunity against L. major if they have been infected with a low dose of parasites (8). Thus, we had to exclude that skin-homing macrophages eliminate the parasites after Curdlan contact shortly after infection. A milder course of disease would be the artificial consequence. In line with published data (31, 33), we were able to demonstrate that macrophages express Dectin-1 in the presence or absence of parasites (Figures S6A–D in Supplementary Material). However, Curdlan activation does not induce leishmanicidal mechanisms resulting in the elimination of L. major parasites (Figure S7 in Supplementary Material). In line with this finding, we were able to show that Curdlan-treated and control BALB/c mice show the same parasite burden at the site of infection during the early phase (days 3 and 14) of experimental leishmaniasis (Figure 3D). At day 40, after infection the beneficial effect of Curdlan treatment (the effect of Curdlan treatment on the parasite load at day 80 after infection, is shown in Figure S5 in Supplementary Material) is slightly indicated but not significant (Figure 3D).

In conclusion, we can demonstrate that the presence of Curdlan at the time of intradermal infection does not induce antigen-unspecific side effects but triggers the modulation of an adaptive T-cell response of BALB/c mice in a Dectin-1-dependent manner: they develop a resistant phenotype.

The rapid clearance of L. major parasites after a high-dose reinfection and the positive DTH response of protected BALB/c mice (Figures 3B,C) supports the hypothesis that an adaptive Th1 response was generated. To assess that possibility, Leishmania-specific IgG subtypes, Th1- and Th2-indicating cytokines, and selected transcription factors were characterized.

Protective immunity to L. major infection does not depend on the production of specific Abs (78). However, L. major-specific IgG responses are an indication for the ability of the host organism to mount an antigen-specific immune reaction. In experimental leishmaniasis, BALB/c mice mount a Th2 response and produce predominantly IgG₁, whereas resistant C57BL/6 mice develop a Th1 response and an increased production of IgG_2c Abs (3, 79). An increase in parasite-specific IgG_2c is therefore an indicator for an ongoing protective Th1 response. In mouse strains with the Igh1-a allele, like BALB/c, the IgG_2c gene is deleted (80). Thus, we determined Leishmania-specific IgG₁ and IgG_2a subsets in sera of BALB/c mice that had been infected in the presence or absence of Curdlan. First, we analyzed mice that had not been treated with Curdlan. C57BL/6 and BALB/c mice generated Leishmania-specific IgG₁ 25 days after infection (Figure 4A). In contrast, C57BL/6 mice produced a significantly higher amount of IgG_2a isotypes compared to susceptible BALB/c mice (Figure 4B). The infection in the presence of Curdlan led to an increase in Leishmania-specific IgG_2a in BALB/c mice (Figure 4C) that also provoked a higher IgG_2a/IgG₁ ratio of Leishmania-specific Abs (Figure 4D). Based on these data, we conclude that Curdlan treatment might dampen the development of a disease-promoting Th2 response in BALB/c mice.

In our study, we used a high-dose infection model and could demonstrate that Curdlan application results in protective immunity in BALB/c mice (Figure 3). It is important to mention that BALB/c mice do not necessarily develop a severe course of disease. In low-dose infection models, BALB/c mice develop a resistant phenotype, accompanied by the polarization toward a Th1-like phenotype (8). Thus, CD4⁺ T cells from BALB/c mice can also differentiate into Th1 cells.

To address the question, whether protected BALB/c mice show a Th1 or Th2 profile, CD4⁺ T cells were purified from SDLNs and analyzed by qRT-PCR. It is commonly accepted that activated CD4⁺ T cells that receive IL-4 signaling upregulate GATA-binding protein 3 (GATA3) and become capable of producing Th2 cytokines (81, 82). Thus, GATA3 is regarded as an inducer for Th2-cell differentiation (83).

We measured the relative expression of GATA3 in CD4⁺ T cells isolated from SDLNs of infected BALB/c mice. These qRT-PCR data revealed that Curdlan treatment results in a dampened expression of GATA3 at day 10 (Figure S8A in Supplementary Material), which is significantly reduced at day 28 (Figure S8C in Supplementary Material) after infection compared to control animals. Furthermore, four out of five Curdlan-treated BALB/c mice also showed a clear reduction in IL-4 expression within the pool of CD4⁺ T cells (Figures S8B,D in Supplementary Material). Thus, a cutaneous application of Curdlan seems to dampen an early IL-4 production within SDLNs. Surprisingly, CD4⁺ T cells isolated from SDLN of Curdlan-treated mice showed a transient trend toward a lower expression of the key transcription factor T-bet, known to define Th1 cells (84), and of the Th1 cytokine IFN-γ at day 10 after infection (Figures S8E,F in Supplementary Material). In contrast, at day 28 the expression of T-bet (Figure S8 in Supplementary Material) but not of IFN-γ (Figure S8H in Supplementary Material) was slightly increased in CD4⁺ T cells of Curdlan-treated BALB/c mice.

A rich body of literature described the fact that Th-cell responses are characterized by the balance of Th1- and Th2-associated cytokines and not by the relative amount of single cytokines such as IFN-γ and IL-4 (85–90). Consequently, we calculated the Th1/Th2 ratio based on the relative expression levels of GATA3, IL-4, T-bet, and IFN-γ at days 10 and 28 after infection. As shown in Figures 5A,B, cutaneous Curdlan application was associated with a modulation of the IFN-/IL-4 ratios at day 10 and 28, indicating a shift toward a pronounced Th1 and a dampened Th2 response at day 28 after infection (Figure 5C). The IFN-/IL-4 ratios of BALB/c mice, infected in the absence of Curdlan showed no modification (Figure 5D). In line with this findings, the ratios of Th1-, Th2-driving transcription factors (T-bet/GATA3) was not substantial different at day 10 but significant enhanced at day 28 (Figures 5E,F), if mice had been treated with Curdlan. Additionally, we were able to demonstrate that this Curdlan-induced Th1 polarization increased from day 10 to day 28 after infection (Figure 5G). T-bet/GATA3ratios of BALB/c mice, infected in the absence of Curdlan showed no modification (Figure 5H). The Th1 polarization was also accompanied by a pronounced IL-17A mRNA expression of CD4⁺ T cells (data not shown).

As shown earlier, Dectin-1⁺ DCs expand at the site of infection and within SDLNs after infection with Leishmania parasites (Figure 2). A dermal exposure to Curdlan can protect BALB/c mice from severe leishmaniasis and induces T cell-mediated immune responses protecting from a high-dose reinfection (Figure 3). Cutaneous DCs are known to be crucial for the generation of adaptive immunity. These professional APCs capture parasites or parasite-derived antigens and are able to migrate to SDLNs for subsequent antigen presentation to T cells (14). To accomplish these tasks, DCs undergo a maturation program accompanied by the expression of surface molecules pivotal for migration and of costimulatory molecules (17). Thus, we wanted to find out whether Dectin-1⁺ DCs get infected by parasites and if these parasite-harboring Dectin-1⁺ DCs undergo maturation after Curdlan treatment.

BMDCs were cocultured with CFSE-labeled promastigote parasites. This allows the differentiation of infected versus non-infected BMDCs subsets simultaneously (Figure 6A) (57). With this method, we could demonstrate that L. major⁺ BMDCs express a higher level of Dectin-1 compared to uninfected DCs (Figures 6B,C). The frequency of Dectin-1-positive BMDCs was also significantly higher in the infected BMDC population compared to BMDCs that were negative for L. major parasites (Figure 6D). Comparable to BMDCs, BMDMs show also a moderate increase in the frequency of Dectin-1⁺ cells and Dectin-1 expression intensity after exposition to L. major parasites (Figure S6 in Supplementary Material). This would suggest Dectin-1-mediated host pathogen interactions. Thus, we addressed the question whether pathogen-derived carbohydrates can interact with C-type lectins and especially with Dectin-1.

The CLRs DC-SIGN (CD209) and the mannose receptor C-type 1 (CD206) (91) have been shown to bind Leishmania antigens. However, it is unknown whether Dectin-1 recognizes L. major-derived molecules. With the help of selected CLR-Fc fusion proteins, we were able to demonstrate an interaction of L. major parasites with CLEC-9 and MGL-1 that recognizes f-actin (92) or galactose, respectively (93) (Figure S9 in Supplementary Material). However, a binding of Dectin-1-Fc fusion proteins to parasite structures could not be observed (Figure S9 in Supplementary Material). Consequently, even though myeloid cells such as macrophages and DCs are positive for Dectin-1 and show a higher Dectin-1 expression after parasite exposition, Dectin-1 alone is not crucial for the uptake of L. major parasites, because the parasites are negative for the Dectin-1 ligand. The fact that Dectin-1^−/− phagocytes can still take up parasites supports that conclusion (31).

L. major parasites can be detected preferentially in BMDCs showing an immature phenotype (Figures 7A,B) and do not induce a maturation of infected cells (data not shown). Thus, parasites seem to inhibit the maturation of immature and Dectin-1^high DCs and thereby the subsequent migration to the SDLN (94, 95). The incubation of infected BMDCs with Curdlan is accompanied by a strong release of TNF-α and IL-6 and a maturation of infected BMDCs (Figure 7C; Figure S10 in Supplementary Material). This is of special interest, because TNF-α and IL-6 are known to be crucial for DC maturation and subsequent priming of T cells (17, 18). Based on the cytokine microenvironment, infected and non-infected Curdlan-treated BMDCs express high levels of CD86 and thus present a mature phenotype (Figures 7D,E). The stimulation of infected BMDCs with Curdlan also induces a strong release of T-cell growth factor IL-2 (Figure S10 in Supplementary Material). In association with the massive induction of costimulatory molecules such as CD86, Curdlan seems to represent an ideal adjuvant for efficient priming of L. major-specific T cells (96). In line with other published data, we showed that Curdlan activates BMDC maturation in a Dectin-1-specific manner (Figure S11 in Supplementary Material) (97, 98).

Additionally, we addressed the question whether Dectin-1 can be detected on Curdlan-activated BMDCs. In this context, we were able to demonstrate that immature BMDCs show in general a higher Dectin-1 expression compared to mature BMDCs (Figure S12A in Supplementary Material), and the detection of surface Dectin-1 is diminished after Curdlan stimulation (Figures S12B,E in Supplementary Material). The population of mature BMDCs, that is positive for L. major parasites, shows also a reduction in the Dectin-1 expression after stimulation with Curdlan (compare Q2, Figure S13 in Supplementary Material). BMDCs that have been exposed to parasites but do not harbor parasites, show no substantial reduction in Dectin-1 expression (compare Q2, Figure S13 in Supplementary Material).

In conclusion, we were able to demonstrate that Curdlan activation results in a maturation of infected BMDCs in a Dectin-1-specific manner that in turn might be crucial for the efficient priming of antigen-specific CD4⁺ T cells.

The adjuvant effect of Dectin-1 ligation in T-cell immunity is undisputed, and it has been proven that Curdlan can polarize T-cell responses (99, 100). In line with these data, it has been shown that anti-Dectin-1 stimulation and the application of microparticulate β-glucan conjugates can potentiate an ovalbumin-specific CD4⁺ T-cell response (101, 102). It was also described that Curdlan treatment can switch a Th2 to a Th1 response in tumor immunology (103). Last but not least, Dectin-1 ligation is discussed to be an important checkpoint in Th17-mediated immunity (28, 34, 38, 97, 104). However, the potential of Curdlan to accelerate the expansion of L. major-specific CD4⁺ T cells has not been described yet. Thus, we isolated CD4⁺ T cells from SDLN of infected BALB/c mice 10 days after infection. BMDCs were pulse-infected with L. major parasites and incubated in the presence or absence of Curdlan. The incubation of CD4⁺ T cells with Curdlan-activated BMDCs resulted in a mild CD4⁺ T cell proliferation (Figures 8A,D). Proliferation of CD4⁺ T cells was substantially increased if BMDCs were pulsed with L. major parasites (Figures 8B,D). A pronounced enhancement of CD4⁺ T cell proliferation was achieved by activating L. major-infected BMDCs with Curdlan (Figures 8C,D). Consequently, Curdlan is capable of triggering the antigen-specific expansion of CD4⁺ T cells by infected BMDCs in a Dectin-1 specific manner (Figure S14 in Supplementary Material). Our data are in line with other experimental models showing that a Curdlan-induced priming of T cells depends on the Dectin-1 pathway (29). Curdlan also enhances the priming of antigen-specific T-cells by BMDCs exposed to Leishmania antigens (Figure S15 in Supplementary Material).

Supernatants of restimulated T cells were characterized by Multiplex analysis, to determine the expanded Th-cell subset. We could show that Curdlan treatment results in a 10-fold pronounced release of IFN-γ, IL-17, and IL-22 compared to control mice (Figure S16 in Supplementary Material). Thus, we conclude that Curdlan treatment induces a Th1 response in BALB/c mice that overlaps with a co-expression of Th17 cytokines (Figure S16 in Supplementary Material). Of note, the release of Th2 cytokines is not substantially modified (Figure S16 in Supplementary Material).

In combination with Curdlan, whole parasites might be used as a successful live “vaccine.” This immunization protocol protects BALB/c mice upon challenge with a high-dose reinfection from severe leishmaniasis (compare Figure 3). Consequently, protected BALB/c mice must have developed an adaptive T-cell memory response capable of controlling and eliminating L. major parasites (75). We investigated whether vaccination of BALB/c mice with L. major antigens (SLA) in combination with Curdlan might also be a promising approach. We tested the adjuvant potential of Curdlan by vaccinating mice intradermally with a mixture of SLA and Curdlan. 28 days later, mice were challenged by a high dose (3 × 10⁶) of L. major parasites into the contralateral footpad. Control mice that had received SLA in the absence of Curdlan developed a severe course of leishmaniasis that correlated with massive ulceration (compare Figure 3). These control groups were not protected, and the experiment had to be terminated at day 42 according to the animal health and welfare practices (Figures 9A,B).

These results again confirm the already published data that the simple administration of antigens does not induce protection of leishmaniasis in BALB/c mice (105). However, some BALB/c mice were partially protected after vaccination with SLA combination with Curdlan (Figure 9A). These mice developed a milder course of disease. Even though footpad swelling was still detectable, the ulceration of lesions was milder and substantially delayed (Figure 9B). Of note, a complete protection as achieved with living parasites could not be induced. The mice with delayed but ensuing mild ulcerations and constant footpad swelling were sacrificed during the monitoring period according to the guidelines for the care and use of experimental animals (Figure 9B). All analyzed lesions showing a delayed ulceration also exhibited a high parasite load (RPU > 1 × 10⁸).