Published online before print March 23, 2004
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* Imperial College London, Faculty of Medicine, Department of Immunology, United Kingdom;
Institute of Pathology, University Hospital Freiburg, Germany; and
Max-Planck-Institute for Immunbiology, Freiburg, Germany
1Correspondence: Imperial College London, Faculty of Medicine, Division of Investigative Science, Department of Immunology, Norfolk Place, London, W2 1PG, United Kingdom. E-mail: i.muller{at}imperial.ac.uk
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: TLR4 • IL-12Rß2 • Leishmania
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The innate immune system is essential for host defense; it senses the presence of potentially pathogenic-invading microorganisms, and the contribution of Toll-like receptors (TLRs) to this response is increasingly recognized [19 20 21 22 ]. TLRs recognize pathogen-associated molecular patterns (PAMPs), which are characteristic of various groups of pathogens. TLRs are expressed on many cell types, including macrophages and dendritic cells (DCs), and the activation of these receptors initiates signaling pathways that result in the up-regulation of costimulatory molecules, the production of proinflammatory cytokines, and anti-microbial compounds [23 ]. Mammalian TLRs are a family of at least 10 pattern-recognition receptors, all of which share an intracellular domain, Toll/interleukin-1 receptor (IL-1R), and signal through the MyD88 adaptor protein [24 25 26 ]. TLRs recognize a wide spectrum of ligands including lipopolysaccarides (LPS), bacterial lipoproteins, proteins, and nucleic acids [22 ]. In contrast to the ample evidence of recognition of bacterial, fungal, and viral PAMPs, very little work has been done on the role of TLRs in recognition of parasites. Recently, glycosylphosphatidylinositol anchors and glycoinositolphospholipids from Trypanosoma cruzi were shown to activate TLR2 [27 ], and lipophosphoglycan (LPG) from L. major was reported to activate natural killer (NK) cells through TLR2 [28 ]. Indirect evidence for the involvement of TLRs in the host response to infections with Leishmania parasites originates from studies with genetically resistant mice lacking the MyD88 adaptor protein; these mice develop progressive lesions and a polarized Th2 response [29 , 30 ].
Our previous work has shown that C57BL/10ScCr mice, which carry a null mutation in tlr4 corresponding to a 74-kb genomic deletion, removing all three exons [31
], were unable to heal L. major infections [32
]. In contrast, TLR4-competent controls (C57BL/10ScSn) could resolve the cutaneous lesions and control parasite growth [32
]. However, it cannot be concluded from our previous study that the observed, nonhealing L. major infections in the C57BL/10ScCr mice are a result of the lack of TLR4, as these mice not only lack tlr4, they also exhibit an impaired interferon-
(IFN-) response to bacteria and parasites [33
34
35
]. The defective IFN- response in the TLR4-deficient C57BL/10ScCr mice is caused by an inability to respond to IL-12 [36
]. The genetic defect underlying this inability to respond to IL-12 was shown to be a result of a point mutation in the IL-12Rß2 gene, resulting in a defective IL-12Rß2 subunit [37
]. However, they are not completely defective in their ability to produce IFN-, as they were capable of producing this cytokine when stimulated with anti-CD3 monoclonal antibodies (mAb) or the T cell mitogen concanavalin A [32
33
34
35
].
To answer whether TLR4 contributes to innate and adaptive immune responses to L. major, we used the progenitor strain of the C57BL/10ScCr mice, C57BL/10/ScNCr, which carries an identical null mutation in tlr4 but has an intact IL-12 responsiveness, a functional IL-12Rß2 chain, and normal IFN- production [36
]. In the present study, we investigated the course of L. major infection in two different strains of TLR4-deficient mice, C57BL/10ScCr and C57BL10/ScNCr, and compared it with infections in the TLR4-competent C57BL/10ScSn mice. The results of our study show that although the second defect in the IL-12Rß2 is undoubtedly a major factor in the nonhealing L. major infections, the lack of TLR4 in the IL-12Rß2 intact mice results in increased parasite growth in the innate phase of infection and a delayed healing of L. major infection.
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Six- to 12-week-old animals of both sexes were used in this study.
Parasites and infection
L. major LV39 (MRHO/SU/59/P strain) was maintained in a virulent state by monthly passage in mice [40
]. For infections, 2 x 106 stationary-phase parasites were injected subcutaneously (s.c.) into the hind footpad. The lesions were measured by determining the increase in the footpad thickness compared with the uninfected contralateral footpad using a dial gauge calliper (Kröplin Schnelltaster, Schlüchtern, Germany).
Determination of parasite load
The number of living L. major parasites in infected tissues was determined using the parasite-limiting dilution assay [40
, 41
]. Briefly, serial dilutions of the footpad homogenate were distributed in replicate wells, and the plates were incubated at 26°C. After 10–14 days, the assay was read microscopically, and the number of viable parasites in the tissue was determined as described [40
].
Macrophages
Bone marrow was obtained by flushing the femurs of naïve mice. Bone marrow precursor cells were cultured as described previously [32
] in hydrophobic Teflon bags (Biofolie 25, Heraeus, Hanau, Germany) in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal calf serum, 5% horse serum, and the supernatant of L929 fibroblasts at a final concentration of 15% (v/v) as a source of colony-stimulating factors, which drive the cell proliferation toward a pure population of bone marrow-derived macrophages. After 9–10 days of culture, macrophages were harvested, and 5 x 105 cells ml–1 were plated and stimulated in the presence or in the absence of 20 U ml–1 IFN-, 200 U ml–1 tumor necrosis factor 
(TNF-), 20 U ml–1 IL-4, 1 µg ml–1 LPS, and 25 x 105 ml–1 L. major parasites.
Nitrite determination
NO2– accumulation was used as an indicator of NO production and measured by using the Griess reagent [9
, 42
]. Culture supernatants were collected after 48 h, and equal volumes of macrophage culture supernatants and Griess reagent [1% sulphanilamide/0.1% N-(1-naphthyl)ethylenediamine dihydrochloride/2.5% H3PO4] were mixed and incubated for 10 min at room temperature. Absorbance was measured at 540 nm in a microscope plate reader (Molecular Devices, Ismaning, Germany). Nitrite concentration was determined using NaNO2 as standard.
Determination of arginase activity
Arginase activity was measured in macrophage lysates as described previously [12
]. Briefly, cells were lysed with 100 µl 0.1% Triton X-100. After 30 min on a shaker, 100 µl 25 mM Tris-HCl was added. To 100 µl of this lysate, 10 µl 10 mM MnCl2 was added, and the enzyme was activated by heating for 10 min at 56°C. Arginine hydrolysis was conducted by incubating the lysate with 100 µl 0.5 M L-arginine (pH 9.7) at 37°C for 15–20 min. The reaction was stopped with 800 µl H2SO4 (96%)/H3PO4 (85%)/H2O (1/3/7, v/v/v). The urea concentration was measured at 540 nm after addition of 40 µl -isonitrosopropiophenone (dissolved in 100% ethanol) followed by heating at 95°C for 30 min. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol urea per min.
Cytokine measurements
Cells (5x106 ml–1) from the lymph nodes draining the lesions of individual mice were restimulated with 1 x 106 ml–1 L. major promastigotes. Forty-eight hours later, the culture supernatants were harvested, and cytokines were determined by enzyme-linked immunosorbent assay (ELISA) according to the suppliers’ protocols. Detection limits were 1 U ml–1 for IFN- and 7 pg ml–1 for IL-4.
Cell recruitment to the site of parasite inoculation
The footpads of ScSn, ScN, and ScCr mice were homogenized in Tenbroeck glass homogenizers 1 day after infection. A Ficoll gradient was performed to eliminate debris, and the recovered viable cell suspension was washed and preincubated with 1 µg rat anti-mouse mAb CD32/CD16 (Fc receptor for immunoglobulin G II/III), PharMingen, San Diego, CA] in a final volume of 50 µl in staining buffer (phosphate-buffered saline, Sigma Chemical Co., St. Louis, MO) supplemented with 3% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY) and 0.1% NaN3 (Sigma Chemical Co.) for 5 min to reduce nonspecific binding. Rat anti-mouse CD49b mAb (clone DX5, PharMingen) and rat anti-mouse CD11b mAb (clone M1/70, PharMingen) were directly added to the cells in a final volume of 1 µl and incubated for 20 min on ice. Cells were washed and resuspended in 200 µl fluorescein-activated cell sorter medium, cell-surface markers were determined by flow cytometry (EPICS XL, Coulter, Luton, UK), and the data were analyzed using the Expo32 software (Beckman Coulter, Fullerton, CA).
Histology
Following formaldehyde fixation, the footpads underwent an EDTA decalcification; they then were cut in two parts following their longitudinal axis and embedded in paraffin using standard protocols. Histological sections (5 µm-thick) were prepared of the entire cut surfaces from both parts of each footpad and stained with haematoxylin and eosin. The histological evaluation was performed under a light microscope using magnifications from ten- to 400-fold.
Statistical analyses
Experimental results were analyzed with PRISMTM version 2.0 (GraphPad, San Diego, CA) using a Student’s t-test, and differences were considered statistically significant at P < 0.05.
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Figure 1. (A) Parasite load in the footpads of ScSn and ScCr mice during the course of L. major infection. Groups of ScSn and ScCr mice (n=4) were infected with 2 x 106 L. major in the footpads. At the time-points indicated, the numbers of viable parasites were determined in the infected footpads by limiting-dilution assay (LDA). The values represent the average of four individual mice per group ± SEM. Data show the results of one representative experiment out of four. N.D., Not detectable; *, not applicable. (B) Lesion development in ScSn, ScN, and ScCr mice during the course of L. major infection. Groups of ScSn, ScN, and ScCr mice (n=4) were infected with 2 x 106 L. major in the footpads. The values represent the average of four individual mice per group per time-point ± SEM. Data show the results of one representative experiment out of three. *, P < 0.05.
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Figure 2. (A) Parasite load in the footpads of ScSn, ScN, and ScCr mice 1 and 28 days postinfection. Groups of ScSn, ScN, and ScCr mice (n=4) were infected with 2 x 106 L. major in the footpads. At the time-points indicated, the numbers of viable parasites were determined in the infected footpads by LDA. The values represent the average of four individual mice per group ± SEM. Data show the results of one representative experiment out of four. (B) Activation of NO synthesis in macrophages from ScSn, ScN, and ScCr mice. Bone marrow macrophages (5x105 ml–1) from naïve, TLR4-competent ScSn control (solid bars), ScN (open bars), and ScCr (hatched bars) mice were cultured in the presence and/or in the absence of 25 x 105 ml–1 L. major promastigotes and the indicated combinations of LPS (1 µg ml–1), IFN- (20 U ml–1), and TNF- (200 U ml–1). Culture supernatants were collected after 48 h and tested for NO2–. Data show the results of one representative experiment out of four. (C) Induction of arginase activity in macrophages from ScSn, ScN, and ScCr mice. Bone marrow macrophages (5x105 ml–1) from naïve, TLR4-competent ScSn control (solid bars), ScN (open bars), and ScCr (hatched bars) mice were cultured in the presence and/or in the absence of 25 x 105 ml–1 L. major promastigotes and IL-4 (20 U ml–1). After 48 h of culture, the macrophages were lysed, and the arginase activity was measured.
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by macrophages derived from the TLR mutant strains. Stimulation with LPS, IFN-, and L. major resulted in a marked reduction of the NO production by the TLR4-deficient macrophages from ScN and ScCr mice as compared with those elicited in macrophages derived from TLR4-competent ScSn mice (Fig. 2B)
. It is important to note that the NO pathway is not defective in macrophages from both TLR4-deficient strains of mice, as stimulation with IFN-, TNF-, and L. major resulted in a strong production of NO in all three strains of mice (Fig. 2B)
. Determination of the arginase activity showed that infection of macrophages from all three strains of mice with L. major parasites did not result in the induction of arginase (Fig. 2C) , whereas activation with IL-4 alone resulted in marginally higher arginase activity in macrophages from the TLR4-deficient mice (Fig. 2C) . It is interesting that activation of parasitized macrophages with IL-4 clearly resulted in the induction of a more pronounced arginase activity in macrophages from both strains of TLR4-deficient mice (Fig. 2C) . These results demonstrate that in the absence of TLR4-mediated signaling, alternatively activated L. major-infected macrophages express higher arginase activity, which could contribute to the enhanced parasite growth by promoting the polyamine synthesis.
Antigen-specific Th1 and Th2 cytokine production
It has been shown previously that ScCr mice display an impaired IFN- response to bacteria and parasites [32
33
34
35
]. To determine whether the absence of TLR4 signaling has an influence on the Th phenotype, we measured the production of antigen-specific IFN- and IL-4 by the lymph node cells from all three strains of L. major-infected mice (Fig. 3
). There was a slightly higher level of IFN- produced by the cells from the ScN mice compared with the control ScSn mice (P>0.05). As expected, the lymph node cells from the ScCr mice produced low levels of IFN- and high levels of IL-4. No IL-4 was detectable by ELISA in the supernatants from the ScSn and the ScN mice. These results suggest that in contrast to the defect in the IL-12Rß2 chain, the absence of TLR4 signaling does notinfluence the production of antigen-specific IFN- or IL-4 by the lymph node cells.
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Figure 3. Antigen-specific production of IFN- and IL-4. Groups of ScSn, ScN, and ScCr mice (n=4) were infected with 2 x 106 L. major in the footpads. Four weeks post infection, the lymph nodes draining the lesions were harvested and stimulated with L. major. Forty-eight hours later, the supernatants were harvested and tested for their content of cytokines by ELISA. The values represent the average of four individual mice per group ± SEM. Data show the results of one representative experiment out of two.
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Figure 4. Histological analysis of the footpads from ScSn, ScN, and ScCr mice. Groups of ScSn, ScN, and ScCr mice (n=3–5) were infected with 2 x 106 L. major in the footpads. Ten hours later, the footpads were processed as described in Materials and Methods. (A) Footpad section from a naïve ScSn mouse. Hematoxylin and eosin (H&E): original primary magnification, x200. (B) Footpad section from L. major-infected ScSn mouse 10 h postinfection. H&E: original primary magnification, x200. A mild mixed infiltration of mononuclear cells and granulocytes is seen in the corium. (C) Footpad section from L. major-infected ScN mouse 10 h postinfection. H&E: original primary magnification, x200. A marked confluent infiltration of granulocytes can be seen in the corium.
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Table 1. Semiquantitative Histopathological Analysis of Footpads from Naive and L. major-Infected ScSn, ScN, and ScCr Mice
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Table 2. Lack of TLR4 and TLR4/IL-12Rß2 Signaling Alters the Cellular Infiltration at the Site of Infection
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Figure 5. Parasite load in the footpads of ScSn, ScN, and TCr5/ScN mice. Groups of ScSn, ScN, and TCr5/ScN mice (n=4) were infected with 2 x 106 L. major in the footpads. At the time-points indicated, the numbers of viable parasites were determined in the infected footpads by LDA. The values represent the average of four individual mice ± SEM.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Thus, the results we obtained with L. major-infected SnCr mice confirm a role for IL-12 in the development of Th1 responses and in resistance to L. major infections. Similar results have been reported recently using L. major-infected IL-12Rß2 knockout (KO) mice on the C57BL/6 background; these mice developed large lesions, which ulcerated earlier than those of susceptible BALB/c mice, and displayed a Th2 cytokine profile and were also severely compromised in their ability to secrete IFN-, a finding consistent with a lack of Th1 responses [51
]. Although the findings discussed above confirm a role for IL-12 in L. major infections, they do not answer whether the lack of TLR4 affects L. major infections. To answer this question, we used the progenitor strain of the ScCr mice, the ScN mice, which also lack TLR4 but have a normal ability to respond to IL-12 [36 ]. In the present study, we show that the lack of TLR4 in IL-12Rß2 intact C57BL/10ScNCr mice resulted in an increased permissiveness for parasite growth during the innate and the adaptive phase of the immune response and in significantly delayed healing of the cutaneous lesions as compared with the TLR4-competent control mice. The enhanced intracellular survival and multiplication of Leishmania parasites in their main host cells, the macrophages, correlated with increased arginase activity and reduced NO synthesis in bone marrow macrophages from TLR4-deficient mice. It is well documented that the induction of the iNOS pathway and the synthesis of NO are essential for parasite killing [7 , 9 , 49 ]; thus, reduced NO levels will result in less-efficient parasite killing. In contrast, the growth of the intracellular parasites is dependent on the availability of polyamines, and arginase catalyses the hydrolysis of L-arginine into urea and L-ornithine, an amino acid essential for the synthesis of polyamines used for the proliferation of Leishmania parasites [10 , 11 ]. Several studies in mice indicate that the balance between arginase and iNOS represents an important mechanism in controlling macrophage function [12 , 55 , 56 ]. Based on the reduced synthesis of NO and the increased arginase activity in alternatively activated L. major-infected macrophages from TLR4-deficient mice, it is tempting to speculate that TLR4 signaling influences the regulation of the L-arginine metabolism in infected macrophages. We conclude from the increased parasite growth in the absence of TLR4 in L. major-infected mice that signaling through this innate recognition receptor contributes to the control of parasite growth in the early as well as in the chronic phase of L. major infection. The underlying molecular mechanisms are not yet identified and are a major focus of our current research. The ligand activating TLR4 after L. major infection has not yet been identified, and as TLR4 not only interacts with pathogen-associated PAMPs but can also recognize endogenous ligands such as heat shock proteins [57 ] and components of the extracellular matrix [58 ], infection-induced endogenous host factors could also play a role. Furthermore, it is also possible that cooperation between different TLRs or between TLR4 and parasite-specific receptors is required [59 , 60 ]. Independently of the nature of the ligand-activating TLR4 after L. major infection in vivo, the use of TLR4-transgenic TCr5 mice demonstrated unequivocally that TLR4 contributes to the efficient control of Leishmania growth in vivo.
Immediately after infection with L. major, a local inflammatory process is initiated, and the very early events after parasite entry, the recruitment of cells into the site of lesion, may be essential for the outcome of disease. Indeed, investigation of the early inflammatory response at the cutaneous site of infection in genetically resistant C57BL/6 and in susceptible BALB/c mice has shown that the early, inflammatory response in nonhealer mice is characterized by more pronounced features of acute inflammation and by a more pronounced recruitment of PMNs as well as by a higher proportion of infected PMNs [13 , 61 , 62 ]. The cellular recruitment to the site of parasite inoculation early after L. major infection was strikingly different between TLR4-competent controls and the two strains of TLR4 mutant mice used in the present study. The marked infiltrate of granulocytes in the lesions of TLR4-deficient and TLR4/IL-12Rß2 double-deficient mice could contribute to the observed differences in healing and parasites clearance. PMNs have been associated with efficient innate defense mechanisms in different experimental models of bacterial, fungal, and parasitic infections, and neutrophils are able to phagocytose Leishmania promastigotes [63 ]. Although PMNs are considered to be primary effector cells, which are involved in the first line of defense, they can also enhance parasite survival [62 , 63 ]. Neutrophils have a very short lifespan; they spend only 6–10 h in circulation before they die. It is interesting that internalization of living L. major parasites can delay spontaneous apoptosis of neutrophil granulocytes in vivo and extend the lifespan of these cells [13 , 18 ]. The delayed apopotosis of infected neutrophils could result in increased survival of the intracellular parasites in the first few days of infection. Macrophages, the main host cells of Leishmania, are not present at high numbers at the very early time of infection; they are recruited later into the lesions. Therefore, phagocytosis of Leishmania by granulocytes at the site of lesion, combined with the ability of Leishmania parasites to delay the apoptosis of infected PMNs [13 ], could represent an escape mechanism resulting in increased parasite survival in the initial phase of infection. Moreover, the uptake of apoptotic cells does not activate antimicrobial effector mechanism in macrophages [64 ]. Therefore, as suggested recently [13 ], it is possible that the uptake of Leishmania-infected neutrophils could help the parasites to enter the macrophages silently and avoid the activation of defense strategies in macrophages. Furthermore, a role for the early wave of infiltrating PMNs in the instruction of Th2 responses in BALB/c mice has been reported [62 ], and depletion of PMNs shortly before L. major infection of BALB/c mice prevented the early IL-4 burst, decreased Th2 responses, and resulted in partial inhibition of lesion progression and parasite growth [62 ]. Thus, the increased recruitment of PMNs to the site of parasite entry could contribute to the enhanced multiplication of Leishmania parasites in TLR4-deficient mice.
In contrast to the breadth of existing knowledge on the regulation and modulation of adaptive immune response to L. major, the understanding of the innate immune responses and the contribution of TLR-mediated functions to these responses are still very limited. Indirect evidence for a role of TLRs in L. major infections was obtained recently by using MyD88 KO mice on a genetically resistant C57BL/6 background. L. major activates IL-1 in macrophages through a MyD88-dependent pathway in vitro [65
], and MyD88 KO mice failed to develop a protective response after L. major infection in vivo; they displayed large, cutaneous lesions and a polarized Th2 response, similarly to susceptible BALB/c mice [29
, 30
]. These results show clearly that the MyD88 protein is crucial for the efficient clearance of L. major parasites and for the induction of protective Th1 responses [29
, 30
], but they do not provide direct evidence that TLRs are activated in vivo after L. major infection. The cytoplasmic MyD88 adaptor protein is one of the key mediators of the signal transduction for all TLRs; however, it is not exclusive for TLR signaling pathways, and it is also common to IL-1R, IL-18, and IL-1R-associated protein kinase signaling pathways.
It is interesting that LPG, which has no counterpart in other eukaryotes, has recently been identified as a ligand for TLR2 [28
29
30
]. LPG, a complex glycophospholipid, is one of the major surface molecules of Leishmania parasites [66
, 67
] and contributes to the virulence of the parasites [68
, 69
]. LPG expression is developmentally regulated, and stage-specific changes occur during metacyclogenesis, when the parasite differentiates from a procyclic promastigote to an infective metacyclic promastigote [70
71
72
]. LPG from L. major has been shown in an in vitro transfection system to activate TLR2 signaling [30
]. LPG is recognized by TLR2 expressed on human NK cells, and the LPG-TLR2 interaction results in an up-regulation of TLR2 message and cell-surface expression and the activation of NK cells to secrete IFN- and TNF- [28
]. The extent of the NK cells activation was dependent on the developmental changes that occur in the carbohydrate residues of the phosphosaccharide repeat units of the LPG molecule during metacyclogenesis, and LPG purified from metacyclic promastigotes was a more potent stimulator than LPG from procyclic parasites [28
].
In summary, our results presented here confirm the important role of IL-12R-mediated signals for the development of protective immunity, and furthermore, they identify TLR4 as a contributor in the complex host parasite interplay in the early phase of L. major infection. It is likely that complex organisms such as Leishmania parasites express several PAMPs and that a cooperation of different innate recognition receptors is required for the effective host defense against these eukaryotic parasites. The present study indicates that TLR4 is one of the innate recognition receptors that contributes to the innate response against L. major infection.
Received October 17, 2003; revised January 29, 2004; accepted February 9, 2004.
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-hydroxy-L-arginine controls the growths of Leishmania inside macrophages J. Exp. Med. 193,777-7838 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice Immunity 6,541-549[CrossRef][Medline]/ß) and type 2 nitric oxide synthase regulate the innate immune response to a protozoan parasite Immunity 8,77-87[CrossRef][Medline] Interferon mediates Propionibacterium acnes-induced hypersensitivity to lipopolysaccharide in mice Infect. Immun. 60,1994-2001 production induced by gram-negative bacteria in mice J. Exp. Med. 181,953-960 Interferon production in endotoxin-responder and -nonresponder mice during infection Infect. Immun. 59,3484-3491 expression in macrophages through a MyD88-dependent pathway Microbes Infect. 4,763-771[CrossRef][Medline]This article has been cited by other articles:
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