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,1
,1,2
,4
* Institute for Experimental Dermatology, University of Münster, Münster, Germany;
Research Group Infection Genetics, Department of Experimental Mouse Genetics;
Junior Research Group Immunodynamics, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany; and
Department of Dermatology, University of Münster, Münster, Germany
4Correspondence: Research Group Infection Genetics, Department of Experimental Mouse Genetics, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. E-mail: andreas.lengeling{at}helmholtz-hzi.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,25-dihydroxyvitamin D3 (1,25(OH)2D3) can selectively suppress key functions of interferon-gamma (IFN-
) activated macrophages. To further explore this mechanism for its relevance in vivo, we investigated an infection model that crucially depends on the function of IFN- activated macrophages, the infection with the intracellular protozoan Leishmania major. 1,25(OH)2D3 treatment of L. major infected macrophages demonstrated a vitamin D receptor (Vdr) dependent inhibition of macrophage killing activity. Further analysis showed that this was a result of decreased production of nitric oxide by 1,25(OH)2D3-treated macrophages due to Vdr-dependent up-regulation of arginase 1 expression, which overrides NO production by Nos2. When analyzing the course of infection in vivo, we found that Vdr-knockout (Vdr-KO) mice were more resistant to L. major infection than their wild-type littermates. This result is in agreement with an inhibitory influence of 1,25(OH)2D3 on the macrophage mediated host defense. Further investigation showed that Vdr-KO mice developed an unaltered T helper cell type 1 (Th1) response on infection as indicated by normal production of IFN- by CD4+ and CD8+ T cells. Therefore, we propose that the absence of 1,25(OH)2D3-mediated inhibition of macrophage microbicidal activity in Vdr-KO mice results in increased resistance to Leishmania infection.
Key Words: interferon-gamma • host resistance • macrophage • knockout mice • parasite
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,25-DIHYDROXYVITAMIN D3(1,25(OH)2D3), the active metabolite of vitamin D3 is known to act as an important regulator of calcium and bone metabolism (1)
but can also influence the immune response (2)
. The vitamin D receptor (Vdr), which mediates the biological effects of 1,25(OH)2D3 and belongs to the superfamily of nuclear receptors, is expressed on different cells of the immune system, including cells of the myeloid and lymphoid lineage (3
, 4)
. The impact of 1,25(OH)2D3 on adaptive immune responses is known to be immunosuppressive. Within a variety of experimental Th1 cell-mediated autoimmune diseases, including experimental autoimmune encephalomyelitis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, and type I diabetes, 1,25(OH)2D3 can prevent or ameliorate the disease (5
6
7
8
9
10
11)
. In addition, Vdr agonists are the most used topical agents in the treatment of psoriasis, an inflammatory skin disease, indicating the potential of 1,25(OH)2D3 and its analogues as therapeutics for immunointervention and treatment of inflammatory diseases (12
13
14
15)
. The ability of 1,25(OH)2D3 to suppress inflammation and to promote tolerance has been linked with its capacity to modulate dendritic cell (DC) and T cell functions. It has been reported that the steroid can suppress T cell proliferation (16)
and decrease the production of the Th1 cytokines IL-2, IFN-, and TNF-, leading to the inhibition of Th1 cell development in vitro (17)
. In addition, 1,25(OH)2D3 and its analogs were shown to inhibit DC differentiation and maturation and to impair the capacity of DCs to induce alloreactive T cell activation (18
19
20)
.
While the effects of 1,25(OH)2D3 on T cell activation and differentiation have been studied extensively, much less is known about its effects on innate immune responses. Early reports have demonstrated that activated macrophages can produce 1,25(OH)2D3 (21
, 22)
. However, the biological significance of this macrophage response in vivo has not been investigated so far. Recently, we demonstrated that 1,25(OH)2D3 is a potent suppressor of IFN--mediated macrophage activation (23)
. 1,25(OH)2D3 was shown to suppress the expression of numerous IFN--inducible genes and inhibit listericidal activity by suppressing the generation of phagocyte-derived reactive oxygen. This deactivation of IFN--stimulated macrophages was strictly depending on a functional Vdr and required a synergistic induction of Vdr expression and accumulation of the protein in the cell nucleus (23)
. This led us to suggest that the production of 1,25(OH)2D3 by IFN--activated macrophages might be an important negative feedback mechanism to control innate and inflammatory responses of activated macrophages (23)
.
To investigate the role of the suppressive effect of 1,25(OH)2D3 on macrophage functions in vivo, we used an animal model where activated macrophages play a decisive role as IFN--dependent effector cells in host defense, the infection with the protozoan parasite Leishmania major. In experimental leishmaniasis macrophages play a dual role. First, they are the major host cells that take up and harbor the obligatory intracellular parasite at the site of infection (24)
. Secondly, they present the only cell type that is capable to efficiently eliminate even a high-load of the parasite (25)
. However, the resolution of disease and the elimination of the pathogen crucially depend on the activation of macrophages by IFN-. This cytokine stems from L. major specific Th1/Tc1 cells, which are generated in resistant mice, while in susceptible mice L. major specific Th2 cells are unable to activate killing activity of macrophages (25
26
27)
. The decisive step in control of experimental Leishmaniasis is the continuous activation of macrophages by IFN-, which leads to induction of the type 2 nitric oxide synthase (Nos2) and therefore generation of the highly leishmanicidal nitric oxide (NO) (28
, 29)
. The IFN- induced production of NO is not only crucial for the direct leishmanicidal activity of macrophages but also triggers the IL-12 signaling cascade that leads to further IFN- production by natural killer cells (30)
. Consequently, mice deficient either in the interferon- gene (Ifng) or the inducible NO synthetase gene (Nos2) are highly susceptible toward Leishmania infection (30
31
32)
. The production of NO can be counter regulated by expression of arginase 1 in alternatively activated macrophages, whereas impairment of this mechanism can be correlated with delayed disease progression after Leishmania infection in vivo (33
, 34)
.
Given the suppressive effects of 1,25(OH)2D3 on IFN- activation and a substantial role of IFN- for host defense against L. major, we investigated a potential role of the steroid in this infection model. We exposed wild-type and Vdr-KO macrophages to 1,25(OH)2D3 and IFN- and analyzed their capability to eliminate the parasite. To further investigate the molecular mechanisms involved in macrophage-mediated Leishmania killing and Vdr-dependent modulation of leishmanicidal activity, we quantified expression of inducible Nos2 and arginase 1 (Arg1) and production of nitric oxide and arginase activity. The relevance of our findings in vitro was further analyzed in vivo by employing the Leismania infection model. The course of experimental leishmaniasis in Vdr-mutant and wild-type mice was investigated by measurement of footpad swelling, quantification of parasite loads in cutaneous lesions using the limiting dilution assay (LDA) and characterization of cytokine production by CD4+ and CD8+ T cells from infected Vdr-KO and wild-type mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was purchased from Pepro Tech (London, UK), recombinant murine macrophage colony stimulating factor (M-CSF) and 1,25(OH)2D3 were from Sigma-Aldrich (Taufkirchen, Germany), recombinant murine IL-4 from BD Pharmingen (Heidelberg, Germany) and recombinant murine GM-CSF was from R&D Systems (Minneapolis, MN, USA). IFN- was diluted in PBS and used at a final concentration of 500 U/ml. 1,25(OH)2D3 was initially dissolved in ethanol and added to the cell culture medium at a dilution of 1: 1000 (final concentrations = 0.04, 0.4, 4, 40 nM), whereas the controls included the respective amount of ethanol.
Animals
C57BL/6 mice were purchased from Harlan (Borchen, Germany) and bred in the specific pathogen-free (SPF) animal facilities of the HZI. Vdrtm1Rge knockout mice (35)
were obtained from Reinhold Erben (Vienna, Austria) and maintained on a C57BL/6 background. All mice were maintained on a calcium-rich diet (2% calcium, 1.25% phosphorus, 20% lactose; ssniff, Soest, Germany). For infection experiments, mice were matched for age and sex (females, 8–12 wk old). On infection with L. major, mice were kept under sterile conditions in microisolator cages in the biosafety level 2 facilities of the Münster University (Department of Dermatology) with unlimited access to food and water according to federal animal protection regulations. All animal studies were reviewed and approved by the local authorities (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit and Bezirksregierung Münster).
Parasites
L. major (WHO nomenclature MHOM/IL/81/FE/BNI) were cultivated in Schneider’s Drosophila Medium supplemented with 10% FCS, 2% human urine, 2% glutamine, and 1% penicillin/streptomycin (Leishmania medium) as described previously (36)
. Soluble leishmania antigen (sLmAg) was prepared by five freeze and thaw cycles in PBS.
Preparation of bone marrow-derived macrophages (BMDMs)
BMDMs were differentiated in vitro from bone marrow stem cell progenitors. Briefly, mice were killed and the femurs were removed, cleaned of tissue and flushed with HBSS. Erythrocytes were depleted by osmotic shock and cells were washed with HBSS, collected by centrifugation, and cultured in DMEM (Invitrogen, Karlsruhe, Germany) containing 2 mM glutamine (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 100 mg/ml Penicillin/Streptomycin (Biochrom, Berlin, Germany) and 10% heat inactivated FCS (Biochrom). This macrophage medium was supplemented with M-CSF at a concentration of 50 ng/ml and bone marrow cells were cultured for 6 days.
Phagocytosis assay
L. major were incubated in 10% mouse serum for complement opsonization and labeled with the fluorescent dye 5-(and 6)-carboxyfluoresceindiacetat succinimidylester (CFDA-SE, Molecular Probes, Leiden, The Netherlands) as described previously (37)
. Parasites were added in a ratio of 5:1 to macrophages and cells (1x106 cells/ml) were seeded into 24 well tissue culture plates (Greiner, Frickenhausen, Germany) and incubated for 4 h to determine the initial rate of phagocytosis. Thereafter, cultures were washed to remove extracellular parasites and macrophages were detached by incubation in 5 mM EDTA for 30 min and washed three times. To distinguish between parasites adhering to macrophages and internalized Leishmania we additionally used a rat monoclonal antibody (8E7) against L. major promastigotes and amastigotes and a phycoerythrin (PE)-labeled secondary antibody as described previously (38)
. After washing, cells were analyzed by flow cytometry and the percentage of cells that had engulfed labeled parasites as well as the percentage of cells with adhering parasites were determined using CellQuest Pro software from Becton Dickinson (Heidelberg, Germany).
Killing rate of macrophages
Macrophages were seeded into LabTekR culture chamber slides (Nunc, Naperville, IL, USA) at a number of 5 x 104 cells per chamber. Macrophages were primed with IFN- (500 U/ml) and/or 1,25(OH)2D3 (40 nM) or left untreated for 24 h. L. major were opsonized in 10% mouse serum for 30 min, washed and added at a ratio of 5:1 to the cultured macrophages. After 4 h at 37°C extracellular parasites were removed by repeated washing and slides were stained using DiffQuick (Dade Boehring, Marburg Germany). The efficacy of phagocytosis was evaluated at 4 h after infection by counting the number of amastigote L. major parasites in 100 macrophages. The antileishmanial activity was assessed by monitoring the number of amastigote L. major parasites in 100 macrophages 24 h after infection.
RT-PCR quantification of gene expression
For RT-PCR, 1 µg of RNA was reverse transcribed using random hexamers (Amersham, Freiburg, Germany) and Superscript II RNase H– Reverse Transcriptase (Invitrogen). Real-time quantitative PCR was performed on an Applied Biosystems RT-PCR System PRISM T 7000 using the Brilliant SYBR Green QPCR Core Reagent Kit (Stratagene, La Jolla, CA, USA). Expression was normalized to the housekeeping genes ribosomal protein S9 (Rps9) or to glyceralaldehyde-3-phosphate dehydrogenase (Gapdh), when appropriate expression was also normalized to unstimulated controls. Oligonucleotides used for amplification were for Nos2: 5'-CGCTTTGGCACGGACGAGA-3', 5'- AGGAAGGCAGCGGGCACAT-3', for Arg1: 5'-GGTCCACCCTGACCTATGTG-3', 5'-GCAAGCCAATGTACACGATG-3', for Gapdh: 5'-GTCCACCAGCCTGTTGCTGTAG-3', 5'-CCCACTCTTCCACCTTCGATG-3' and for Rps9: 5'-CTGGACGAGGGCAAGATGAAGC-3', 5'-TGACGTTGGCGGATGAGCACA-3'.
For analysis of Arg1 and Nos2 expression in L. major infected footpads the expression levels of both genes were normalized to Emr1 transcript levels. Emr1 encodes the F4/80 protein, which is a seven trans-membrane G-protein coupled receptor routinely used as a marker for murine macrophages. Oligonucleotides used for Emr1 amplification were 5'-CTGTAACCGGATGGCAAACTTG-3' and 5'-ACACAGCAGGAAGGTGGCTATG-3'.
NO release and inhibition of arginase activity
NO release was measured as concentration of nitrite in the supernatant using Griess reagent as described (39)
. Shortly, macrophages were plated at a density of 4 x 105/ml and left untreated or stimulated with 500 U/ml IFN-, 40, 4, 0.4 and 0.04 nM 1,25(OH)2D3 or corresponding combinations of IFN- and 1,25(OH)2D3 or left untreated. For inhibition of arginase activity 100 µM nor-NOHA (Sigma-Aldrich) was included in the assay. After 24 h L. major was added to one half of cultured cells in a ratio of 5:1 and nitrite concentration was measured 24 h later. Macrophages were harvested and total RNA was isolated or cells were used for determination of arginase activity.
Arginase assay
After removal of the supernatants for measurement of NO release, cells were suspended in sonication buffer (50 mM, Tris-HCl, pH 7.4; 0.1 mM EDTA; 0.1 mM EGTA) supplemented with complete Mini Proteinase Inhibitor tablets (Roche, Mannheim, Germany) and sonicated (2x20 s). Lysates were then diluted 1/10 and 50 µl of diluted cell lysate was mixed with 50 µl manganese buffer (50 mM Tris-HCl pH 7.4, 10 mM MnCl2) and heated at 60°C for 10 min. 50 µl L-Arginine (0.5 M, pH 9.7, Sigma-Aldrich) was added and samples were incubated at 37°C for 1 h. After the addition of 400 µl stopping solution (H2SO4:H3PO4:H2O = 1:3:6) and 25 µl a-Isonitrosopropiophenone (9% in ethanol, Sigma-Aldrich), samples were incubated at 95°C for exactly 45 min. The OD550 was then determined using an ELISA plate reader. In parallel, the protein concentration of the lysates was determined using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA) according to the manufacturers instructions. Arginase activity was determined as the amount of urea produced (using a urea standard curve) and normalized to the amount of total protein.
In vivo infections
Experimental leishmaniasis was initiated by subcutaneous application of 2 x 107 promastigotes (stationary phase) of L. major in 50 µl PBS into the left hind footpad (at least 5 mice per set of experiment). Footpad thickness was measured with a metric caliper. Footpads, spleens and livers from mice were harvested after 2, 5, and 8 wk of infection for determination of cytokine profile and limiting dilution assay (LDA). Footpads were also harvested 5 wk p.i. for RNA preparation and determination of Arg1 and Nos2 expression. The tissue was grinded in liquid nitrogen and total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instructions.
Limiting dilution assay
Parasite numbers in footpads, spleen, and liver were determined 8 wk after infection by limiting dilution assay (LDA) (36
, 40)
by using Leishmania medium instead of slant blood agar. Briefly, organs were removed aseptically and homogenized in 5 ml Leishmania medium. Serial dilutions were carried out in quadruples (100 µl culture volume each) using 96-well tissue plates. After culture for 1 wk, the highest dilution yielding growth of viable parasites was determined using a phase contrast microscope.
Cytokine assay with L. major-specific CD4+ and CD8+ T cells
At 2, 5, and 8 wk after infection with L. major, mice were euthanized and spleens were aseptically removed. A single cell suspension was prepared and CD4+ and CD8+ T cells were generated as described (36)
. Briefly, cells were purified by passage over a nylon wool column and were subsequently depleted of contaminating cells using MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and an automated magnetic cell sorter (autoMACS; Miltenyi Biotec) according to the manufacturers instructions.
Bone marrow-derived DCs were generated from C57BL/6J mice as described previously (38)
. Briefly, bone marrow was isolated and cultured in the presence of IL-4 and GM-CSF for 6 days. DCs (1x106 cells/ml) were incubated with sLmAg equivalent to 5 x 106 L. major for 48 h. For assessment of cytokine secretion, DC and CD4+ T cells were mixed at a ratio of 1:5 and cultured in RPMI1640 plus 2 mM glutamine, 50 µM mercaptoethanol and 10% FCS for 48 h. Culture supernatants were assayed by ELISA (BD Pharmingen) for IL-4 (detection limit < 14 pg/ml) and IFN- (detection limit < 2.5 pg/ml) according to the manufacturers instructions.
Statistical analysis
Data are presented as mean ± SEM unless indicated otherwise. Statistical analysis was performed using unpaired Student’s t test. Differences were considered to be statistically significant for P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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activation of infected macrophages. We had previously demonstrated that 1,25(OH)2D3 is a potent suppressor of IFN--mediated macrophage activation (23)
. To investigate the effect of 1,25(OH)2D3 on effector functions of macrophages after infection with L. major, we analyzed the capacity of BMDMs from Vdr-KO and wild-type mice to phagocytose and kill L. major as well as to produce NO in the absence and presence of 1,25(OH)2D3.
Phagocytosis of L. major by macrophages from Vdr mutant and wild-type mice
Fluorescently labeled L. major were added in a 5-fold excess to cultured macrophages for 4 h, and the rate of phagocytosis was determined by flow cytometry. To distinguish between adhering and internalized parasites, we additionally stained with an antileishmanial antibody. The number of adhering parasites was found to be low in all experiments (<5%, data not shown). Comparable phagocytosis rates were observed in macrophages from wild-type and Vdr-KO mice (Fig. 1
). 66% of wild-type and 70% of Vdr-KO macrophages were infected. The presence of 40 nM (Fig. 1)
or lower concentrations of 1,25(OH)2D3 (data not shown) did not result in an alteration of phagocytic capacity in macrophages of both genotypes (68% in wild-type and 69% in Vdr-KO mice). We repeated the experiment four times and could not measure any difference in phagocytic rates. Thus, we did not detect an influence of 1,25(OH)2D3 on the phagocytic capacity of macrophages.
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Killing activity by macrophages from Vdr mutant and wild-type mice
To investigate the potential of macrophages from Vdr mutant and wild-type mice to eliminate intracellular parasites in the presence or absence of 1,25(OH)2D3, we determined numbers of intracellular parasites and infection rates of macrophages (Fig. 2
, and data not shown). As already demonstrated by flow cytometry, we observed no differences in initial phagocytosis rates after 4 h (data not shown). Killing of intracellular parasites was induced by activation with 500 U/ml of IFN- and monitored 24 h after addition of L. major. As expected, activation of macrophages with IFN- resulted in a decrease of parasites/macrophage as well as in the percentages of infected macrophages. Addition of 1,25(OH)2D3 to nonactivated macrophages had no effect on both parameters. However, when cells were cultured in the presence of 40 nM 1,25(OH)2D3 and IFN-, killing of intracellular parasites was completely inhibited in wild-type macrophages. In contrast, Vdr-KO macrophages showed no inhibition of IFN- mediated killing activity (Fig. 2)
. Thus, Vdr-mediated signaling by 1,25(OH)2D3 suppressed IFN- induced killing of L. major.
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Release of NO by macrophages from Vdr-KO and wild-type mice
Intracellular killing of L. major by macrophages requires production of NO. Therefore, we compared NO secretion by macrophages from Vdr-KO and wild-type mice on stimulation with IFN- and infection with L. major in the absence or presence of different 1,25(OH)2D3 concentrations (Fig. 3
). As expected, NO production by macrophages was not detectable in the absence of L. major parasites (data not shown). When IFN--activated cells from both Vdr mutant and wild-type mice were incubated with L. major, considerable amounts of NO could be detected. In line with the results from the killing assays, 1,25(OH)2D3 dose-dependently inhibited IFN--induced NO generation in L. major infected wild-type cells. In contrast, inhibition of NO production was not detectable in Vdr-KO macrophages (Fig. 3)
. The generation of NO in murine macrophages is differentially regulated by the enzymes inducible nitric oxide synthase (Nos2) and arginase 1 (Arg1), which both competitively use L-arginine as a substrate (for review see 41
). Inhibition of NO production by 1,25(OH)2D3 could, therefore, result either from decreased expression of Nos2 or from enhanced expression of Arg1. When we quantified mRNA expression of Nos2, we found it to be induced by IFN- as expected (Fig. 4
A). However, addition of 1,25(OH)2D3 did not result in inhibition but enhancement of Nos2 expression, which cannot be correlated with the decreased NO production (Fig. 4A
). Intriguingly, the expression of Arg1 was clearly elevated in the simultaneous presence of 1,25(OH)2D3 and IFN-. In contrast, Arg1 was not induced when macrophages were exposed to IFN- or 1,25(OH)2D3 alone (Fig. 4B
). This response was completely abrogated in Vdr-KO macrophages (Fig. 4B
). This was not due to a general deficiency in Arg1 induction in Vdr-KO macrophages, as stimulation with interleukin-4 (IL-4) or double treatment with IFN- and IL-4 induced comparable levels of Arg1 expression in wild-type and Vdr-KO macrophages (data not shown). Similarly, wild-type macrophages infected with L. major and treated with INF- and 1,25(OH)2D3 showed induction of Arg1 and Nos2 expression, whereas in Leishmania infected Vdr-KO macrophages the up-regulation of both genes was not observed (Supplemental Fig. 1). Titration of 1,25(OH)2D3 concentration to IFN- stimulated Vdr-KO and wild-type macrophages demonstrated a concentration and Vdr-dependent induction of Nos2 and Arg1 expression (Fig. 4C
). To investigate if this induction of Arg1 mRNA expression correlates with increased enzymatic activity at the protein level, we quantified arginase activity in Leishmania infected macrophages that were either exposed to IFN- or INF- and 1,25(OH)2D3. Treatment of infected macrophages with IFN- alone did not induce arginase activity above background levels (Fig. 5
A). In contrast, macrophages that were treated with IFN- and 1,25(OH)2D3 clearly showed induction of arginase activity, thus confirming that the up-regulation of Arg1 observed at the transcript level correlates with increased enzymatic activity (Fig. 5A
). These results suggested that the Vdr-dependent induction of Arg1 in the presence of INF- and 1,25(OH)2D3 would override NO production of macrophages. To directly test this hypothesis, we treated Leishmania infected macrophages in the presence of either IFN- or IFN- and 1,25(OH)2D3 with the arginase inhibitor nor-NOHA and quantified NO production. As expected, NO release was diminished in macrophages treated with IFN- and 1,25(OH)2D3 as compared to macrophages that were treated with IFN- alone. However, in the presence of the arginase inhibitor nor-NOHA no differences in NO production of IFN- and IFN-/1,25(OH)2D3 treated macrophages were detected (Fig. 5B
). Taken together these results clearly show that 1,25(OH)2D3 can inhibit leishmanicidal activity in IFN--activated macrophages by up-regulation of Arg1 expression, which antagonizes NO production by Nos2. Since Arg1 acts upstream of Nos2 in NO metabolism, the increased expression of Arg1 is likely to overcome the parallel up-regulation of Nos2, resulting in decreased production of leishmanicidal NO in wild-type macrophages. This response is not functional in Vdr-deficient macrophages, which consequently leads to augmented NO production and increased intracellular killing of Leishmania. Elevated Arg1 expression is a hallmark of alternative macrophage activation induced by IL-4 (42
43
44)
, and impairment of alternative macrophage activation has been demonstrated to be associated with improved macrophage leishmanicidal effector functions and delayed cutaneous Leishmaniasis in macrophage/neutrophil-specific IL-4R-deficient mice (33)
. To investigate whether treatment of macrophages with IFN- and 1,25(OH)2D3 results in development of an alternative activation phenotype we investigated expression of the resistin-like gene (Retnla, previously known as Fizz1), of the chitinase 3-like 3 gene (Chi3l3, previously known as Ym1) and of the mannose receptor gene (Mrc1), which are established marker genes for IL-4-induced alternative activation (45
, 46)
, by real-time RT-PCR. While we were not able to detect induction of Retnla and Chi3l3, Mrc1 expression could be induced by 1,25(OH)2D3 as described previously (47, and data not shown). We, therefore, propose that activation of macrophages by IFN- and 1,25(OH)2D3 leads to a distinct macrophage activation phenotype, similar but not identical to IL-4-induced alternative activation.
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Course of experimental leishmaniasis in Vdr-mutant and wild-type mice
Since we were able to show that 1,25(OH)2D3 inhibits leishmanicidal activity of primary macrophages in vitro, we also analyzed the effects of Vdr signaling on immunity toward infection in vivo. Vdr-KO and wild-type mice were infected with 2 x 107 L. major promastigotes into the hind footpad and disease outcome was monitored. Compared to wild-type littermates, Vdr-KO mice showed a clearly mitigated course of infection (Fig. 6
A). In the first 2 wk after infection, we observed a similar increase in footpad swelling in mice of both genotypes. However, in contrast to wild-type mice, no further progress was found of footpad swelling in Vdr-KO mice until lesions started to resolve 5 wk after infection. At the peak of cutaneous swelling, i.e., 3 wk after infection, Leishmania-induced lesions in wild-type mice were twice as large as in Vdr mutants. Subsequently lesions resolved faster in Vdr-KO mice. In agreement with the development of footpad swelling, wild-type mice harbored more than 300-fold more living parasites in cutaneous lesions as revealed by LDA 8 wk after infection (Fig. 6B
). High numbers of living parasites were found in the lesions of all wild-type mice, while 30% of Vdr-KO mice had already completely eliminated the pathogen. To investigate systemic infection, we also analyzed parasite loads in liver and spleen by LDA. We did not detect living parasites in internal organs of both mutant and wild-type mice (data not shown). Thus, compared to wild-type mice on the C57/BL/6J-resistant genetic background, Vdr mutant mice presented an enhanced capacity to eliminate L. major. To investigate if the increased host resistance of Vdr-KO mice correlates with Arg1 and Nos2 expression in vivo, we compared the expression levels of both genes in L. major infected footpad lesions of Vdr-KO mice and wild-type littermates using real time RT-PCR. The mRNA expression levels of both genes were normalized to Emr1 (F4/80) transcript levels to account for the number of macrophages that had infiltrated the lesions. Five weeks postinfection we found significantly reduced Arg1 expression in lesions from Vdr-KO as compared to Arg1 expression levels in lesions from wild-type mice (Fig. 6C
). Expression levels of Nos2 did not show significant differences between mice of both genotypes. These results show a clear correlation between reduced Arg1 expression in infected footpads from Vdr-KO mice and increased host resistance to Leishmania infection as compared to wild-type controls.
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Cytokine responses of Vdr mutant and wild-type mice infected with L. major
We were able to demonstrate an influence of Vdr-mediated signaling on the leishmanicidal activity of infected macrophages. However, the Vdr is also expressed in CD4+ and CD8+ T cells (4
, 48
, 49)
and 1,25(OH)2D3 has been reported to inhibit Th1 cell differentiation (17)
. Therefore, absence of Vdr-mediated signaling in Vdr-KO mice may result in an intensified Th1 response, which may consequently lead to increased resistance in Vdr-KO mice. To investigate whether the more favorable course of infection in Vdr mutant mice was associated with a change of the L. major-specific T cell response, we compared the cytokine production by CD4+ and CD8+ T cells from infected Vdr-KO and wild-type mice after in vitro restimulation with L. major antigen primed DCs. Both groups of mice showed a L. major specific cytokine pattern typical for a Th1 response (Fig. 7
) with high levels of specific IFN- and barely detectable levels of IL-4 only measurable 8 wk postinfection. No significant differences in IFN- secretion were detected between CD4+ and CD8+ T cells from infected wild-type and Vdr-KO mice after 5 and 8 wk of infection (Fig. 7)
, and there was a slight tendency of even less IFN- being produced by Vdr-KO CD4+ T cells at 2 wk postinfection. Thus, the increased resistance in Vdr-KO mice does not result from an enhanced Th1 response. Therefore, we propose that the increased resistance of Vdr-KO mice to L. major infection results from an augmented killing capacity of macrophages due to the absence of 1,25(OH)2D3 mediated inhibition of leishmanicidal activity in these mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-mediated activation of macrophages and their subsequent enhanced capacity to destroy internalized parasites. In this study, we demonstrate that 1,25(OH)2D3 effectively inhibits the leishmanicidal activity of IFN- activated macrophages. The 1,25(OH)2D3 mediated inhibition of the parasite killing capacity strongly correlates with a diminished NO release from IFN- stimulated macrophages. The suppression of NO production and Leishmania killing by 1,25(OH)2D3 was ablated in macrophages from Vdr-KO mice and is, therefore, strictly dependent on a functional vitamin D receptor signaling pathway. In the mouse model, it is generally accepted that NO is the key antimicrobial agent for killing of L. major by IFN- -activated macrophages. It is well established that NO acts as a microbicidal molecule by production of reactive nitrogen species, which then subsequently modify organic molecules (28
, 32
, 50)
. In addition, NO interferes with the microbial cellular metabolism of Leishmania in the host phagosome (29)
. We could show that the enzyme Arg1 is induced in IFN- activated macrophages on simultaneous treatment with 1,25(OH)2D3. As this response is defect in Vdr-KO macrophages, it might add twofold to the increased leishmanial activity in these cells. First, the Nos2 production of NO is not counterbalanced by induction of Arg1 in Vdr-deficient cells. Secondly, arginase 1 expression is known to favor Leishmania proliferation in macrophages, possibly through generation of polyamines, which are essential for parasite growth (29
, 34
, 51)
. A defect in Vdr-signaling might, therefore, additionally suppress Leishmania growth in IFN- activated macrophages by abolishing Arg1 expression and arginine hydrolysis to urea and ornithine that are used for polyamine synthesis.
We have recently described a new mechanism by which 1,25(OH)2D3 suppresses IFN--mediated activation of macrophages (23)
. 1,25(OH)2D3 was shown to selectively inhibit numerous functions of IFN- -activated macrophages. Among these were the suppression of bactericidal activity, phagocyte oxidative burst, and inhibition of gene expression of important mediators of inflammation and host defense (23)
. Macrophages stimulated with IFN- and infected with Listeria monocytogenes were unable to kill the pathogen when simultaneously treated with 1,25(OH)2D3 (23)
. Here, we show that the 1,25(OH)2D3 mediated suppression of microbicidal activity also extends to the capacity to eliminate intracellular parasites. Since mature macrophages that are activated by IFN- exhibit an increased capacity to actively synthesize 1,25(OH)2D3, we have previously suggested a negative autocrine feedback mechanism acting to control inflammatory responses of these cells (23)
. This mechanism includes a synergistic induction of Vdr expression in macrophages stimulated with IFN- in the presence of 1,25(OH)2D3. When we investigated disease progression in Vdr-deficient mice infected with L. major, Vdr-KO mice showed significantly enhanced containment of the parasites as compared to their wild-type littermates. This was reflected by reduced footpad swellings and lower footpad parasite loads in the Vdr mutant mice. We hypothesize that this is a direct result of an enhanced macrophage killing activity in Vdr-KO mice. The main mechanism seems to be a deficient 1,25(OH)2D3-mediated up-regulation of Arg1 in Vdr mutant mice, even so we cannot exclude that increased oxidative burst might play an additional role (23)
. Previously, it has been shown that under certain conditions Vdr-deficiency has an influence on T cell responses in vivo and that treatment with 1,25(OH)2D3 can ameliorate Th1-driven diseases (6
, 9
, 10)
. In contrast to this data, it has been hypothesized that ablation of Vdr function might alter the Th1/Th2 balance by impairing Th1 cell responses and enhancing the production of Th2 cytokines (52)
. However, Wittke and colleagues who demonstrated that Vdr-deficient mice fail to develop experimental allergic asthma in a mouse model of airway inflammation found that production of IL-4 was reduced while IFN- was not altered in Vdr-KO mice despite higher serum levels in IgE as compared to wild-type controls (53)
. Because polarized Th1 and Th2 cell responses have a tremendous influence on the outcome of Leishmania infection in resistant and susceptible mice, we compared the production of the cytokines IL-4 and IFN- by CD4+ T cells and IFN- production of CD8+ T cells from infected Vdr-KO and wild-type mice after in vitro stimulation with L. major antigen primed DCs. No differences in cytokine profiles were found between both groups of mice, demonstrating that Vdr-KO mice neither displayed enhanced Th1 nor elevated Th2 responses in this infection model. This strongly supports our hypothesis that the increased resistance of Vdr-KO mice to L. major infection is mainly mediated by continuous activation of macrophages by IFN- in the absence of 1,25(OH)2D3 mediated suppression and consequently enhanced production of the highly leishmanicidal NO.
Polymorphisms in the human VDR gene have been associated with increased resistance or susceptibility to a number of infectious diseases. Case-control studies suggested links between certain alleles of the VDR gene and susceptibility to tuberculosis, leprosy, dengue fever, and hepatitis B virus-induced chronic hepatitis (54
55
56
57
58)
. In addition, it has recently been reported that toll-like receptor 2/1 activation in human macrophages leads to up-regulated expression of VDR that in turn induces expression of the antimicrobial peptide cathelicidin, which mediates intracellular killing of Mycobacterium tuberculosis. African-Americans, which are known to have increased susceptibility to tuberculosis, were reported to have low serum levels of 25-hydroxyvitamin D and inefficient induction of cathelicidin expression (59)
. This suggests that differences in human populations to produce vitamin D may contribute to susceptibility or resistance to microbial infection.
We show here that ablation of Vdr function is not necessarily associated with increased host susceptibility toward infection. Instead, Vdr-deficiency was found to increase host resistance toward Leishmania infection by augmenting macrophage defense reactions. We are not aware of any genetic study investigating association of VDR alleles with host defense against parasitic infections. However, our results obtained in the murine system suggest a possible connection of either the vitamin D status or particular VDR alleles that causatively decrease protein activity and VDR function with resistance toward infection with L. major in humans.
Our results obtained from experimental leishmaniasis in mice suggest that inhibition of macrophage effector functions significantly contributes to the immunomodulatory properties of 1,25(OH)2D3 in vivo. Thus, it is tempting to speculate that this mechanism also contributes to the beneficial effects of vitamin D derivates in chronic autoinflammatory processes like rheumatoid arthritis, where macrophages are crucially involved in tissue destruction and disease progression (60)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Current address: Sir William Dunn School of Pathology, University of Oxford, Oxford, Great Britain
3 Current address: Medical University of Innsbruck, Innsbruck, Austria
Received for publication November 28, 2006. Accepted for publication April 19, 2007.
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