TLR9 signaling is essential for the innate NK cellresponse in murine cutaneous leishmaniasis

Jan Liese1, Ulrike Schleicher*1 and Christian Bogdan1,2

1 Institute of Medical Microbiology and Hygiene, Department of Medical Microbiologyand Hygiene, University Clinic of Freiburg, Freiburg, Germany

2 Institute of Clinical Microbiology, Immunology and Hygiene, University Clinic ofErlangen, Erlangen, Germany

Mice deficient for the TLR adaptor molecule MyD88 succumb to a local infection withLeishmania (L.) major. However, the TLR(s) that contribute to the control of thisintracellular parasite remain to be defined. Here, we show that TLR9 was required forthe induction of IL-12 in bone marrow-derived DC by intact L. major parasites orL. major DNA and for the early IFN-c expression and cytotoxicity of NK cells followinginfection with L. major in vivo. During the acute phase of infection TLR9–/– miceexhibited more severe skin lesions and higher parasite burdens than C57BL/6 wild-typecontrols. Although TLR9 deficiency led to a transient increase of IL-4, IL-13 andarginase 1 mRNA and a reduced expression of iNOS at the site of infection and in thedraining lymph nodes, it did not prevent the development of Th1 cells and the ultimateresolution of the infection. We conclude that TLR9 signaling is essential for NK cellactivation, but dispensable for a protective T cell response to L. major in vivo.

Introduction

The protozoan parasite Leishmania (L.) major causeslocalized, papulous or ulcerative skin lesions in a varietyof mammals, including humans and mice. In the mousemodel of cutaneous leishmaniasis, genetically resistantmouse strains (e.g. C57BL/6) develop transient, but self-healing skin swellings at the site of infection, whereasnon-healer strains (e.g. BALB/c) are unable to containthe parasite locally and succumb to a fatal visceraldisease. The control of the parasite, the resolution of thedisease, and the development of long-lasting resistancerequires the production of interleukin (IL)-12 bydendritic cells (DC), the induction and expansion of

type 1 CD4+ T helper cells (Th1) releasing interferon(IFN)-c, the generation of tumor necrosis factor (TNF)and the expression of inducible nitric oxide synthase(iNOS, also termed NOS2) by macrophages [1–5].Conversely, BALB/cmice, or C57BL/6mice lacking IL-12or IFN-c, allow the expansion of IL-4- and IL-13-expressing Th2 cells, which is paralleled by a reducedexpression of iNOS protein and high tissue parasiteburdens [5–9].

NK cells contribute to a protective immune reactionagainst L. major, especially during the early phase ofinfection [10, 11]. The activation of NK cells duringinfection was shown to be triggered by type I interferons(IFN-a/b)and IL-12.ActivatedNKcells in thedrainingLNof L. major-infected mice produced IFN-c and expressedcytolytic activity. The depletion of NK cells or theinhibitionofNKcellactivationbyanti-IFN-a/b treatment,neutralizationorgeneticdeletionof IL-12,or inhibitionofIL-12 signaling in vivo resulted in the loss of early parasitecontainment and of IFN-c production [12–16].

Correspondence: Professor Christian Bogdan, Mikrobiolo-gisches Institut, Universit�tsklinikum Erlangen,Wasserturmstraße 3–5, D-91054 Erlangen, GermanyFax: +49-9131-852-2573e-mail: christian.bogdan@uk-erlangen.de

Received 15/2/07Revised 9/8/07

Accepted 24/9/07

[DOI 10.1002/eji.200737182]

Key words:Leishmania � NK cells� Parasite/protozoaninfection � Toll-like

receptors

Abbreviations: BMDC: bone marrow-derived dendritic cell �LmAg: Leishmania major antigen soluble � SLA: Leishmania majorantigen

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* Additional corresponding author: Dr. Ulrike Schleichere-mail: ulrike.schleicher@uniklinik-freiburg.de

The recognition and signaling events that arenecessary for this early immune activation are largelyunknown. Mice lacking the universal TLR adaptormolecule myeloid differentiation factor 88 (MyD88)on a resistant background lost their ability to control theinfection with L. major [17–19]. This suggests that TLRmight be involved in pathogen recognition and initiationof adequate immune effector functions. Indeed, TLR4-deficient mice had increased parasite burdens through-out the course of infection [20]. However, TLR2/TLR4double deficient mice developed a normal protectiveTh1 response against L. major [18].

In contrast to other TLR both TLR7 and TLR9 areknown to signal in a strictly MyD88-dependent mannerand are endosomally localized [21]. These pathogenrecognition receptors are therefore prone to interactwith phagocytosed parasites such as Leishmania residingin macrophages or dendritic cells. In addition toprokaryotic (bacterial) DNA and oligodeoxynucleotidesthat contain hypomethylated CpG-motifs (CpG ODN),eukaryotic (host cell and parasite) DNA were alsoidentified as TLR9 ligands [21–25]. The possiblerelevance of TLR9 signaling for a protective immuneresponse against Leishmania parasites in vivo was firstsuggested by Zimmermann et al. [26], who found thatL. major-infected BALB/c mice can be protected fromdeveloping fatal visceral disease when treated with CpGODN prior to infection. We recently confirmed theseresults (J. Liese and U. Schleicher, unpublishedobservations).

The findings on L. major-infected MyD88-deficientmice and the considerations summarized aboveprompted us to investigate, whether TLR9 is implicatedin the immune response to L. major. Here, we report thatTLR9–/– mice exhibit a strikingly impaired innate NK cellresponse to L. major, develop more severe skin lesionsthat were paralleled by increased parasite burdens, andshow an altered cytokine expression pattern character-ized by a transient up-regulation of IL-4 and IL-13mRNA. As TLR9 deficiency did not prevent thegeneration of Th1 cells, the mice ultimately resolvedthe cutaneous disease. Thus, TLR9 is essential for theinnate immune response to L. major, but a protectiveT cell response followed by parasite control and clinicalcure of the lesions can occur in the absence of TLR9.

Results

NK cell cytotoxicity and IFN-c release in L. major-infected mice requires TLR9

To assess the role of TLR9 for the innate immuneresponse to a cutaneous infection with L. major,C57BL/6 WT and TLR9–/– mice were infected with

L. major promastigotes. Cytokine mRNA levels weredetermined in the skin lesions and the draining LN at 6and 18 hours after infection. IFN-c mRNA was stronglyand rapidly up-regulated in the draining LN of WTmice,but significantly less so in TLR9–/– mice. The early IFN-cmRNA induction that occurred in the LN was notobserved in the footpad (Fig. 1A).

Since NK cells represent an important source forearly IFN-c in cutaneous leishmaniasis [10, 11, 16, 27],we next investigated, whether there is a difference in theIFN-c expression of NK cells in C57BL/6WTand TLR9–/–

mice. In accordance with previous data [16]CD3–NK1.1+ LN NK cells from L. major-infected WTmice expressed IFN-c protein already after 12 h ofinfection. The number of IFN-c+ cells within the NK cellpopulation was further increased after in vitro restimu-lation with YAC tumor target cells (Fig. 1B). In contrast,LN NK cells from TLR9–/– mice were not induced toexpress IFN-c protein in response to L. major infection invivo and restimulation with YAC cells in vitro. However,LN NK cells from TLR9–/– mice produced IFN-c afterrestimulation with PMA/ionomycin (data not shown),demonstrating that TLR9–/– NK cells are not intrinsicallydefective in the production of IFN-c. In addition, therewas no difference in the numbers of NK cells betweenWT and TLR9–/– mice (data not shown).

After local infection with L. major, NK cells are notonly induced to release IFN-c, but also to expresscytotoxic activity [10, 11]. LN NK cells from L. major-infected (but not from uninfected) WT mice exhibitedprominent lytic activity against YAC tumor target cells.In contrast, LN NK cells from TLR9–/– mice acquired nocytolytic activity after infection with L. major (Fig. 1C).

From these data we conclude that TLR9 is essentialfor the induction of NK cell IFN-c expression andcytotoxicity during the innate phase of L. major infectionin mice.

TLR9 is required for the L. major-inducedexpression of IL-12 by DC

One of the cytokines that is indispensable for theactivation of NK cells during the innate phase ofcutaneous leishmaniasis is IL-12 [12, 13, 15], which isreleased by subpopulations of DC after infection withLeishmania promastigotes or amastigotes [28–30].Thus, we examined whether the expression of IL-12 isreduced in the absence of TLR9. During the first 18 h ofinfection the levels of IL-12p35 mRNA and of IL-12p40mRNA did not change at the site of infection (footpad)or in the draining LN compared to uninfected mice, nordid they differ between C57BL/6 WT and TLR9–/– mice(Fig. 1A). However, as the induction of IL-12 in L. major-infected mice is restricted to a small number of DCduring the first 3 days of infection [30], it might be

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impossible to detect a TLR9-dependent regulation ofIL-12 mRNA using whole organ RNA preparations. Wetherefore performed intracellular cytokine stainingusing unseparated or CD11c+ enriched popliteal LNcells to detect DC expressing IL-12p40/p70 protein ininfected WT versus TLR9–/– mice. Although thistechnique worked well in the case of visceral leishma-niasis after intravenous infection with L. infantum [31],we failed to detect an up-regulation of the number ofIL-12p40/p70+ cells within the CD11b+CD11c+ DCcompartment of draining LN at various time-points aftercutaneous infectionwith L. major as compared to controlmice injected with PBS (data not shown). However,when we analyzed CD11c+CD11b+ bone marrow-derived dendritic cells (BMDC) of WT mice in vitro,both L. major and L. infantum promastigotes caused asignificant increase of the number of IL-12p40/p70+

cells compared to unstimulated cells, which did notoccur in the case of TLR9–/– BMDC (Fig. 2A). A similarobservation was made with L. major amastigotes,although in this case the difference in the number ofIL-12p40/p70+ cells between WT and TLR9–/– BMDC

did not reach the level of significance (Fig. 2A). Underthese in vitro conditions L. infantum was a considerablymore potent inducer of IL-12p40/p70 than L. major(Fig. 2A), which offers an explanation for the difficultyto detect a TLR9-dependent regulation of IL-12p40/p70in L. major-infected mice in vivo.

In order to provide further evidence for a linkbetween TLR9-dependent NK cell activation and IL-12production by DC, we analyzed IL-12p35–/– mice. Wefound that the NK cell IFN-c expression and cytotoxicactivity was equally defective in IL-12p35–/– mice andTLR9–/– mice (Fig. 1C, Fig. 2B, and data not shown).Together, these data support the hypothesis that theearly NK cell response to L. major requires an intactTLR9-IL-12 axis and that the absent NK cell response inTLR9–/– mice is likely to be causally related to a lack ofIL-12 induction by L. major promastigotes.

Nucleic acids are known ligands for TLR9. Therefore,we tested whether CD11c+CD11b+ BMDC express IL-12after exposure to genomic DNA prepared from L. majoror L. infantum (Fig. 2C). Intracellular cytokine stainingclearly revealed a TLR9-dependent induction of

Figure 1. Impaired innate immune response in TLR9–/– mice after infection with L. major. C57BL/6 WT and TLR9 –/– mice wereinfected with 3 � 106 stationary phase L. major promastigote parasites into the hind footpads. (A) Cytokine mRNA expression inpooled draining LN and in the footpad tissue 6 and 18 h after infection using quantitative RT-PCR with assays for the respectivegenes. Results are shown as mean expression levels from three independent experiments (two to five mice per group) with errorbars representing standard deviation (SD; *p<0.01); n.d.: not detectable. (B) LN cells from infected and PBS-treatedWTand TLR9–/–

micewere harvested after 12 h and 1� 106 cellswere incubated in the presence of brefeldin A for 8 h with or without the additionof YAC cells. Cellswere analyzed using intracellular cytokine staining. Percentage of IFN-c+ cells after gating on CD3–NK1.1+ cells isshown. Data are representative of two independent experiments. (C) Draining LN cellswere harvested 36 h after infection and thenumber of CD3–NK1.1+ cells was determined by FACS. Cells were incubated for 4 h with 51Cr-labeled YAC cells at the indicatedratios and specific lysiswas determined. Results are shown from two independent experiments (� SD; *p<0.01 for infectedWT vs.TLR9–/– mice).

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IL-12p40/p70 by Leishmania DNA, with L. infantumDNA being much more potent than L. major DNA. Thesefindings imply that genomic DNA contributes to theTLR9-dependent recognition of Leishmania by dendriticcells.

TLR9–/– mice show an aggravated course ofcutaneous leishmaniasis

The critical role of TLR9 for the innate NK cell and IFN-cresponse to L. major raised the possibility that TLR9 mayalso be required for the generation of an adaptiveimmune response to the parasite, which might lead to analtered course and outcome of infection in the absenceof TLR9. We therefore infected C57BL/6 WT andTLR9–/– mice with L. major promastigotes into the hindfootpads and monitored the development of the skinswelling relative to the footpad thickness beforeinfection (Fig. 3A). TLR9–/– animals developed signifi-cantly more severe lesions in the acute phase of the

infection (days 30–45) compared to WTmice. However,during the late phase of the disease TLR9–/– mice wereable to control the infection similar to WT mice. Wenever observed ulcerated or necrotic skin lesions inTLR9–/– mice. In accordance with the clinical course ofinfection the parasite load in the tissues of TLR9–/– mice(footpad, draining LN and spleen) were only transientlyelevated compared to WT mice (Fig. 3B and data notshown). Thus, TLR9-deficiency leads to an aggravated,but still self-healing course of infection.

Intact IFN-c expression by T cells in L. major-infected TLR9–/– mice

Previously, an enhanced parasite growth and diseaseseverity have been observed in L. major-infected mousestrains, in which the type 1 T helper cell or IFN-cresponse was absent [7, 8] or delayed [15] or in whichthe expression of anti-leishmanial effector moleculeswas impaired or entirely suppressed [4, 14, 32, 33]. In

Figure 2. Expression and function of IL-12 in L. major-infectedmice. (A) BMDC from C57BL/6WTand TLR9–/– micewere stimulatedwith L. major or L. infantum promastigotes (PM) or amastigotes (AM)with a 3:1 parasite:cell ratio or with CpG 1668 (1 lM). After 16 hof stimulation brefeldin A (10 lg/mL) was added for another 6 h. Cells were harvested and surface and intracellular cytokinestaining was performed. The frequency of IL-12p40/p70+ cells after gating on CD11chighCD11b+ cells is shown. Results are meanfrequencies (� SD; *p <0.05) of three independent experiments. (B) BALB/c WTor IL-12p35–/– mice (three to four per group) wereinfected with L. major as in Fig. 1. After 36 h, pooled draining LN cells were incubated for 4 h with 51Cr-labeled YAC cells at theindicated ratios and specific lysis was determined. Results show one representative from two independent experiments (� SD;*p <0.05 for infected WT vs. IL-12p35–/– mice). (C) WT or TLR9–/– BMDC were stimulated as described in panel A with CpG 1668,L. major or L. infantum DNA (10 lg/mL). The frequency of IL-12p40/p70+ cells within the gated CD11chighCD11b+ population wasdetermined using intracellular cytokine staining. The dot blots show the results from one of three independent experiments, withthe mean percentages (� SD) of IL-12p40/p70+ CD11b+CD11c+ cells calculated from all three experiments.

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order to define the mechanistic basis for the enhancedparasite burden in TLR9–/– mice, we analyzed the mRNAexpression of cytokines, iNOS and arginase 1 in footpadtissue and draining LN from L. major-infected WT andTLR9–/– mice using quantitative RT-PCR.

In contrast to the innate phase of infection (day 1,Fig. 1A) the up-regulation and expression level of IFN-cmRNA was indistinguishable between WT and TLR9–/–

mice throughout the acute and late phase of infection.Likewise, the expression of IL-12p35 mRNA, IL-12p40mRNA, TNFmRNA as well as iNOSmRNA, the inductionof which is critically dependent on IFN-c [34, 35], werevery similar in WT and TLR9–/– mice in the footpadlesions and the draining lymph nodes. For these mRNA,significant differences were only detected at single timepoints of infection and only in one of three independentexperiments (Fig. 4A and B, and data not shown).

Additionally, in vitro restimulation of LN cells fromL. major-infected WT and TLR9–/– mice during theclinically acute infection (day 28) with soluble L. major

antigen (SLA) yielded comparable amounts of IFN-c+

CD4+ T cells as determined by intracellular cytokinestaining (Fig. 5). Furthermore, the amounts of IFN-c (asdetected by ELISA) in the supernatants of lymph nodecells restimulated with SLA (WT: 1759 � 463 pg/mL;TLR9–/–: 1103 � 473 pg/mL) or with immobilized anti-CD3 (WT: 3124 � 745 pg/mL, TLR9–/–: 2804 �435 pg/mL; mean � SD of two independent experi-ments) were in the same order of magnitude and notsignificantly different.

In order to rule out that na�ve CD4+ T cells fromTLR9–/– mice have an altered capability to develop intoTh1 or Th2 cells, we isolated splenic CD4+ T cells fromuninfected WT and TLR9–/– mice and differentiatedthem under Th1- or Th2-skewing conditions in vitro (seeMaterials and methods). Subsequent restimulation ofthese cells with anti-CD3 mAb and analysis of cytokineproduction by intracellular cytokine staining or ELISArevealed comparable numbers of IFN-c+ (Th1) or IL-4+

cells (Th2) as well as similar amounts of IL-4 or IFN-cprotein in the culture supernatants for CD4+ Tcells fromboth strains of mice (data not shown). Thus, TLR9–/–

CD4+ Tcells do not exhibit an inherent bias towards Th2cell differentiation.

TLR9–/– mice express increased levels of IL-4,IL-13 and arginase 1 mRNA

In agreement with earlier studies [9, 12, 36] there was arapid up-regulation of IL-4 mRNA in the skin and LN ofC57BL/6 WT mice following infection with L. major(Fig. 4A and B). The same early IL-4 peak occurred inTLR9–/– mice. Importantly, however, the subsequentdecrease of IL-4 mRNA expression, which is character-istic for C57BL/6 WT mice, was considerably delayed inTLR9–/– mice. Thus, we observed higher levels of IL-4mRNA in TLR9–/– mice, starting with the onset of thedevelopment of the skin lesion (days 22–28) in all threeexperiments performed and lasting throughout thecourse of infection (Fig. 4A and B and data not shown).The difference in IL-4 expression between WT andTLR9–/– mice was most prominent and statisticallysignificant in LN and footpad tissue during the acutephase of the disease (days 38–50).

The same observation was made with respect to theexpression of IL-13mRNA (Fig. 4A and B). Both IL-4 andIL-13 are known as strong inducers of arginase 1 inmacrophages [37]. Accordingly, TLR9–/– mice did notonly express higher levels of IL-4 and IL-13, but alsoshowed transiently and significantly increased amountsof arginase 1 mRNA in the skin lesions and drainingpopliteal LN compared to WT mice (Fig. 4A and B).

Figure 3. TLR9–/–mice display larger footpad lesions and higherparasite burdens after infection with L. major. WT and TLR9–/–

mice were infected with 3 � 106 stationary phase L. majorpromastigote parasites in the hind footpads. (A) Clinical courseof infection as determined by lesion size measurements (seeMaterials and methods). Fifteen to sixteen mice per group wereused. One of three independent experiments is shown witherror bars representing SD (*p <0.01). (B) Parasite numbers inthe footpad lesion and in the draining LN were quantified bylimiting dilution analysis at the indicated time points. Three tofour mice per group were individually analyzed. One repre-sentative of three experiments is shown with error bars for SD(significance was indicated (*) when confidence intervals ofsingle mice did not overlap).

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iNOS protein is reduced in the lymph nodes ofTLR9–/– mice

In vitro, arginase 1 can antagonize the translation andenzymatic activity of iNOSwithout affecting the levels ofiNOS mRNA [38, 39]. We therefore performed im-munohistological analyses to examine the expression ofiNOS protein in the LN from WT and TLR9–/– miceduring the acute phase of L. major infection (day 28). LNfrom WT and TLR9–/– mice infected in parallel wereembedded side-by-side in the same specimen molds. Atleast ten sections from each pair of LN were stained foriNOS protein and subsequently evaluated for thenumber, size, and staining intensity of the iNOS-positivecell clusters.

In a total of 44 sections the number of iNOS-positiveclusters per section in WT LN was significantly higherthan in the corresponding TLR9–/– LN (21.8 � 6.2 vs.16.3 � 3.1, mean � SD; p < 0.01). In addition, in themajority of analyzed sections (29 of 44, i.e. 65%) we

Figure 5. Similar production of IFN-c by Th1 cells fromWTandTLR9–/–mice in the acutephase of the disease. Draining LN cellsfrom three mice infected with L. major were isolated at day 28after infection and pooled. Cells were restimulated in vitrowithSLA (final concentration 40 lg/mL), or with concanavalin A(final concentration 5 lg/mL) for 18 h followed by 6 h ofincubation in the presence of brefeldin A (10 lg/mL). Surfaceand intracellular staining was performed and the number ofIFN-c+ cells after gating on CD3+CD4+ cells was determined.Mean values � SD are shown from two independent experi-ments.

Figure 4. TLR9–/– mice exhibit elevated IL-4, IL-13 and arginase 1mRNA levels during the acute phase of an infection with L. major.Total RNA was isolated from single footpads (A) or popliteal draining LN (B) and reverse transcribed. Expression levels weredetermined by using quantitative RT-PCR with assays for the respective cytokine and effector genes, and gene expression wascalculated relative to the expression of the endogenous control gene (HPRT). Results aremean expression levels from three to fourmice per group with error bars indicating SD (*p <0.05 for DCT values). One representative of three independent experiments isshown; n.d.: not detectable.

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found larger and much more intensely stained iNOS-positive clusters in the LN fromWTmice as compared toTLR9–/– mice (Fig. 6). These results are compatible withthe hypothesis that in TLR9–/– mice the increasedarginase 1 expression partially impairs the expression ofiNOS protein, but not of iNOS mRNA (see Fig. 4).Considering the anti-leishmanial activity of iNOS-derived NO these results sufficiently explain thetransiently elevated parasite numbers in the acute phaseof a L. major infection in TLR9–/– mice.

Together, these data demonstrate that an up-regula-tion of Th2 cytokine expression rather than a deficientexpression of Th1 cytokines is associated with theaggravated L. major infection in TLR9–/– mice.

Discussion

Significance of the findings

NK cells from the spleen or liver of mouse cytomegalo-virus-infected WT mice were previously shown tostrongly express IFN-c, which was reduced by 50% inNK cells derived from TLR9–/– mice [40]. Using a mousemodel of visceral leishmaniasis elicited by intravenousinoculation of L. infantum, we recently demonstratedthat TLR9 is also important for the activation of NK cellsduring a non-viral infection [31]. In the present study weinvestigated the role of TLR9 in experimental cutaneousleishmaniasis, which develops after local injection ofL. major and forms an entirely different disease entity.Our analysis led to several new insights into theprocesses underlying the control of L. major by theinnate and adaptive immune system. First, our datashow that both the induction of NK cell cytotoxicity andNK cell IFN-c production that rapidly occurred in WTmice after cutaneous L. major infection strictly requiredTLR9. Second, we demonstrated that in vitro the releaseof IL-12p40/p70 by myeloid (conventional) DC afterinfection with L. major promastigotes [28–30] waslargely dependent on TLR9. We did not succeed in

demonstrating the TLR9 dependency of the productionof IL-12 by lymph node DC derived from L. major-infected mice, because L. major failed to sufficiently up-regulate the expression of IL-12p40/p70. However, theknown NK cell-stimulatory properties of IL-12 [41], thecurrent concepts of DC-NK cell interaction [31, 42, 43],the comparable NK cell activation defect in TLR9–/– andIL-12p35–/– mice (Fig. 1B and C and 2B), and the TLR9-dependent induction of IL-12p40/p70 in BMDC in vitroafter exposure to L. major (Fig. 2A and C) stronglysuggest that the absent NK cell response in L. major-infected TLR9–/– mice results from the impairedIL-12p40/p70 protein production by DC. At this stageof research, however, we cannot formally exclude thatTLR9 governs the expression of additional soluble orsurface-bound molecules of DC, which similar to IL-12might also be essential for the innate NK cell activation,or that TLR9-deficiency affects other cell-types (e.g.macrophages, plasmacytoid dendritic cells) whichpossibly contribute to the activation of NK cells duringL. major infection. Finally, we provide evidence that thedefective NK cell response is associated with anaggravated clinical course of cutaneous leishmaniasis.This is not due to a principal Th1 differentiation defect ora lack of iNOS mRNA induction, but is more likely toreflect the sustained expression of IL-4, IL-13 andarginase 1 and the reduced expression of iNOS protein inthe skin lesion and/or LN of TLR9–/– mice (Fig. 4 and 6).

The role of NK cells and TLR9 for protectionagainst L. major

The results obtained with TLR9–/– mice are in line withprevious reports that showed amore severe, but still self-healing course of L. major infection after depletion of NKcells by antibodies [10, 11]. As IFN-c is a key cytokine forthe development of Th1 cells and the suppression of Th2cell expansion [27, 44, 45], the absent IFN-c expressionby NK cells most likely contributes to the morepronounced expression of IL-4 and IL-13 in TLR9–/–

mice (Fig. 4A and B). However, we do not want to claimthat TLR9 affects T cell cytokine expression onlyindirectly via regulation of NK cell activity. In fact,there is evidence that TLR9 can function as a non-essential costimulatory receptor directly on CD4+ Tcells(reviewed in ref. [46]), which might limit the Th2-typeT cell differentiation in L. major-infected WT mice. IL-4and IL-13 are known inducers of arginase 1 inmacrophages [37], which converts arginine into ureaand ornithine, a precursor of the synthesis of polyamines[38]. Indeed, the increased levels of IL-4 and IL-13paralleled the up-regulation of arginase 1 in TLR9–/–

mice (Fig. 4A and B). Arginase promotes the growth ofintracellular L. major via the generation of ornithine [47,48], but also indirectly via the consumption of arginine,

Figure 6. iNOS protein expression in LN of L. major-infectedWTand TLR9–/– mice (day 28 of infection). Cryostat sections fromWT (A) and TLR9–/– (B) mice were analyzed by anti-iNOSimmunohistology; alkaline phosphatase staining (red), nucleicounterstained with hematoxylin (blue). bar, 100 lm.

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which impairs the production of leishmanicidal NO byiNOS and the expression of iNOS protein in macro-phages [39]. Therefore, the transiently enhancedexpression of arginase 1 [paralleled by a reduction ofiNOS protein (Fig. 6)] probably accounts for theaggravated clinical course of infection in TLR9–/– mice.

The TLR9 dependency of the production ofIL-12p40/p70 by DC after exposure to L. majorpromastigotes sufficiently explains the NK cell activationdefect in TLR9–/– mice. However, we also observed thatL. major amastigotes are able to induce IL-12 expressionby DC TLR9-independently (Fig. 2A). This might be thereason, why the defective innate NK cell response inTLR9–/– mice was not accompanied by an absent orseverely impaired Th1 development and by a progressivecutaneous and visceral disease as it occurs in geneticallynon-healing BALB/c mice or in C57BL/6 mice that carrygene deletions for IL-12 [8], IFN-c [7], IFN-c receptor[49], or iNOS [14, 32].

The only known ligands for TLR9 are viral, bacterialor eukaryotic DNA [21, 50]. Hemozoin, the degradationproduct of heme in mammalian erythrocytes infectedwith Plasmodium parasites, was also reported to signalvia TLR9 [51], but a recent publication attributes thestimulatory capacity to contaminating DNA within thehemozoin preparation [52]. Previous in vitro studiesshowed that DNA isolated from Babesia bovis, Trypano-soma (T.) brucei, and T. cruzi strongly activate myeloidcells for the release of proinflammatory cytokinesincluding IL-12 in a TLR9-dependent manner [22–24,53]. We obtained similar results with genomic DNA fromL. major in this study (Fig. 2C), which suggests that DNAis at least one of the parasite-derived molecules that issensed by the target cells of Leishmania. The intracel-lular localization of TLR9 offers an explanation for theincreased stimulatory capacity of viable Leishmaniacompared to isolated DNA, because the parasite mightact as a “transporter”, which transfers the nucleic acid tothe endosome. However, at this stage of research theexistence of additional leishmanial TLR9 ligands thatactivate myeloid DC in vitro or in vivo cannot beexcluded.

TLR9 and other parasitic diseases

A number of recent studies performed in other infectiousdiseasemodels provided evidence that TLR9 participatesin the generation of a type 1 immune response. Ininfections with the extracellular parasite T. brucei,TLR9–/– mice initially cleared the parasite from theblood, but unlike WT mice developed a second peak ofparasitemia, which was accompanied by transientlyreduced levels of IFN-c and IgG2a in the serum of thesemice. Importantly, however, there was no significantdifference in the mean survival of WT and TLR9–/– mice

[24]. In T. cruzi-infected TLR9–/– mice the number oftrypomastigotes in the bloodstream was increased,which was paralleled by reduced amounts of IL-12p40and IFN-c in the serum and a slightly decreased rate ofsurvival compared to WT mice [53]. In an oral infectionmodel of toxoplasmosis that is characterized by thedevelopment of acute and lethal ileitis driven by a Th1-type immune response, the absence of TLR9 led to ahigher parasite burden, but also to a reduced expressionof IFN-c by CD4+ as well as by CD8+ T cells and acomplete resistance to Toxoplasma gondii-induced ileitis[54]. None of these studies investigated the role of TLR9for the activation and function of NK cells. Based on ourpresent data in mouse cutaneous leishmaniasis and ourprevious results in experimental visceral leishmaniasis[31] we hypothesize that the NK cell activation defect inTLR9–/– mice might contribute to the altered Th1/Th2-balance observed in the other infectious disease models.

In conclusion, we have shown that TLR9 is crucial forthe activation of NK cells during the innate response to acutaneous infection with L. major. As a consequenceL. major-infected TLR9–/– mice developed a morepronounced expression of IL-4, IL-13 and arginase 1along with a reduced expression of iNOS protein and anexacerbated course of infection. Ongoing studies in ourlaboratory address the question whether TLR9 expres-sion and function is linked to the differential suscept-ibility of various mouse strains to L. major.

Materials and methods

Mice

WT C57BL/6 and BALB/c mice were purchased from CharlesRiver Breeding Laboratories (Sulzfeld, Germany). Breedingpairs of C57BL/6 tlr9–/– mice (10th generation backcross toC57BL/6 mice) were kindly provided by Antje Heit andHermann Wagner (Technical University, Munich), breedingpairs of IL-12 p35–/– mice [55] (5th generation backcross toBALB/c) were a gift of G. Alber (University of Leipzig,Germany). All mice used were 6–10 weeks of age and age-matched. They were held under specific pathogen-freeconditions in the animal facilities of our institute. The animalexperiments were approved by the governmental animalwelfare committee.

Parasites and infection

The origin, propagation and preparation of promastigotes oramastigotes of L. major (strain MHOM/IL/81/FEBNI) and ofpromastigotes of L. infantum (strain MHOM/00/98/LUB1)were described before [31, 33, 56]. Mice were infectedbilaterally into the skin of the hind footpads with 3 � 106

stationary phase promastigotes in 50 lL PBS. The footpadswelling was measured with a metric caliper (in mm; Kroeplin,Schl�chtern, Germany) and related to the footpad thickness

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before infection (bilateral infection) as the % increase offootpad thickness. Tissue parasite burden was determined bylimiting dilution analysis [33, 57]. Statistical significance wasassumed when 95% confidence intervals did not overlap.

Genomic DNA of L. major or L. infantum was prepared byproteinase K digestion of promastigotes followed by phenol/chloroform-extraction and ethanol precipitation or by usingthe Blood&Cell Culture DNA Kit (Qiagen, Hilden, Germany).In both cases RNA was removed with DNase-free RNaseA(Invitrogen).

Cytokine mRNA expression analysis

Skin or LN tissue was homogenized in a Mixer Mill MM 200(Retsch, Haan, Germany), total RNA was extracted using theTRIZOL reagent (Invitrogen, Karlsruhe, Germany), andcontaminating genomic DNA was removed with DNase I(DNAfree, Ambion, Austin, USA). Subsequently, 10 lg RNAwere reverse transcribed using the High Capacity cDNAArchive Kit (Applied Biosystems, Darmstadt, Germany). Toassess the amount of target gene levels we used the HT7900quantitative PCR system (Applied Biosystems). Each cDNAwasmeasured in duplicates or triplicates with the following gene-specific assays (TaqMan Gene Expression Assays, AppliedBiosystems): mIFN-c (Mm00801778_m1), mIL-4(Mm00445259_m1), mIL-12a (IL-12p35;Mm00434165_m1), mIL-12b (IL-12p40; Mm00434170_m1),mIL-13 (Mm00434204_m1), mouse iNOS(Mm00440485_m1), and mouse arginase 1(Mm00475988_m1). The gene for mouse hypoxanthineguanine phosphoribosyl transferase-1 (HPRT-1,Mm00446968_m1) was used as endogenous control forcalibration of the mRNA levels. Quantitative analysis wasperformed using the SDS 2.1 software (Applied Biosystems).mRNA levels were calculated by the following formula: relativeexpression = 2–(CT(Target)

–CT(Endogenous control))� f, with f= 104

as an arbitrary factor.

Bone-marrow derived DC and stimulation for IL-12expression in vitro

Immature BMDC were generated in RPMI 1640 culturemedium (supplemented with 2 mM L-glutamine, 23.8 mMNaHCO3,10 mMHEPES, 50 lM2-mercaptoethanol, 10% FCS)in the presence of rmGM-CSF-containing hybridoma super-natant [58]. Briefly, 6 � 106 BM cells were cultured in largecell-culture dishes in RPMI 1640 medium containing 10%(v/v) FCS (PAA Laboratories, Coelbe, Germany). The cellswere substituted twice with fresh medium and GM-CSF duringthe incubation period. After 8 days the nonadherent BMDCwere harvested and stimulated in 24-cm2 tissue culture dishes(6�106 cells/dish) with the respective stimuli and brefeldin Aas detailed in the legend to Fig. 2. Finally, the cells were labeledwith anti-CD11b(clone M1/70)-FITC- and anti-CD11c(cloneHL3)-PE-conjugated antibodies (BD Biosciences, Heidelberg,Germany), fixed and permeabilized with CytopermCytofixJ

(BD Biosciences), washed twice with permeabilization buffer(PBS, 0.5% saponin, 2% FCS), and stained with rat-anti-mIL-12p40/p70(clone C15.6)-APC-conjugated antibody (BDBiosciences) in permeabilization buffer. Finally, FACS analysis

was performed using a FACSCalibur (BD Biosciences) andCellQuestPro Software (BD Biosciences).

Lymph node dendritic cells and IL-12 expression ex vivo

At 3, 12, 24 or 36 h after cutaneous infection with L. majorpromastigotes (3 � 106) the draining popliteal lymph nodeswere removed and single cell suspensions were prepared. Insome experiments, the lymph node cells were enriched forCD11c+ cells by positive selection using anti-CD11c Micro-BeadsJ and MACSJ technology (Miltenyi Biotech, Bergisch-Gladbach, Germany). The cells were treated with brefeldin A(10 lg/mL) for 8 h (in absence or presence of CpG1668[1 lM]) and were then subjected to intracellular IL-12 stainingas described above.

NK cells and IFN-c expression ex vivo

LN cells (1 � 106) from infected or PBS-treated mice werecultured for 8 h in the presence of brefeldin A (10 lg/mL)with or without YAC tumor target cells at a ratio of 1:1, or with50 ng/mL phorbol myristate acetate (PMA; Sigma, Deisenho-fen, Germany) and 750 ng/mL ionomycin (Sigma). The cellswere labeled with anti-CD3(clone 145–2C11)-FITC- and anti-NK1.1-PE-conjugated antibodies, fixed with CytopermCytofixJ

and subjected to intracellular cytokine staining using rat-anti-mIFN-c(clone XMG1.2)-APC-conjugated antibody (BD Bios-ciences) as described above for BMDC.

NK cell cytotoxic activity

The ability of LN NK cells to lyse YAC tumor target cells wasanalyzed in a 51Cr release assay [14]. The number of effectorNK cells (CD3–NK1.1+) was determined by FACS analysis withanti-CD3-APC- and anti-NK1.1-PE-conjugated antibodies (BDBiosciences).

T cell differentiation

Na�ve CD4+ Tcells were isolated from the spleen using a CD4+

T cell isolation Kit (Miltenyi) and stimulated for 3 days withimmobilized anti-CD3 antibody (clone 145–2C11, BD Bio-sciences; culture wells were coated with 5 lg/mL antibody) inthe presence of either rmIL-12 (10 ng/mL; R&D Systems,Wiesbaden-Nordenstadt, Germany) plus anti-IL-4 (10 lg/mL,clone 11B11; BD Biosciences) (Th1-skewed condition) orrmIL-4 (100 ng/mL; R&D Systems) plus anti-IFN-c (1 lg/mL,clone XMG1.2; BD Biosciences) (Th2-skewed condition). Thecells were expandedwith rmIL-2 (5 ng/mL; R&D Systems) andfinally restimulated with immobilized anti-CD3. The fractionof IFN-c- or IL-4-positive cells was determined by intracellularcytokine staining (see above) with rat-anti-mIFN-c-PE- or rat-anti-mIL-4-APC-conjugated antibodies (BD Biosciences), re-spectively. The IFN-c or IL-4 content of cell culture super-natants was determined by capture ELISA (BD Biosciences andR&D Systems).

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In vitro restimulation of total LN cells for IFN-cexpression

Total LN cells were isolated and 2.5 � 105 cells were incubatedwith soluble L. major antigen (SLA, final concentration40 lg/mL; [59]), concanavalin A (5 lg/mL), or in thepresence of plate-bound anti-CD3 mAb (see above). For thedetermination of IFN-c+ cells, brefeldin A was added after18 h of incubation to a final concentration of 10 lg/mL forfurther 6 h. Cell surface staining was performed with anti-CD3-FITC- and anti-CD4(clone GK1.5)-PE-conjugated anti-bodies (BD Biosciences) followed by intracellular cytokinestaining with anti-IFN-c-APC (BD Biosciences) as describedabove. The percentage of IFN-c+ Th1 cells was calculated aftergating on CD3+CD4+ cells. The amount of IFN-c in thesupernatants of restimulated cells was measured with acapture ELISA (BD Biosciences and R&D Systems).

Immunohistology

For the immunohistochemical detection of iNOS, acetone-fixedcryostat sections of LN (5 lm) were incubated with a rabbit-anti-mouse iNOS antiserum [57], followed by biotin-conju-gated F(ab0)2-fragment donkey-anti-rabbit-IgG antibody, alka-line phosphatase-conjugated streptavidin (DakoCytomation,Hamburg, Germany) and a red alkaline-phosphatase substrate(Vector Laboratories, Burlingame, CA) [56]. Sections werecounterstained with Meyer0s hemalaun, mounted with Aqua-texJ (Merck, Darmstadt, Germany) and analyzed by lightmicroscopy (Axioskop 2 plus, Zeiss).

Statistical analysis

Statistical analysis was performed using the two-tailedStudent's t-test with an expected similar variance and p-valuesare shown.

Acknowledgements: We thank Claudia Kurzmann andRosaMammato for excellent technical assistance, AntjeHeit and Hermann Wagner (Technical University,Munich, Germany) for providing TLR9–/– mice and GeorgAlber (University of Leipzig, Germany) for his gift ofIL-12p35–/– mice. This study was supported by grantsfrom the Deutsche Forschungsgemeinschaft to C.B. andU.S. (DFG Bo 996/3–2 and 3–3; SFB 620 project A9).

Conflict of interest: The authors declare no financial orcommercial conflict of interests.

References

1 Bogdan, C., Gessner, A. and R�llinghoff, M., Cytokines in Leishmaniasis: acomplex network of stimulatory and inhibitory interactions. Immunobiology1993. 189: 356–396.

2 Bogdan, C. and R�llinghoff, M., The immune response to Leishmania:mechanisms of parasite control and evasion. Int. J. Parasitol. 1998. 28:121–134.

3 Solbach, W. and Laskay, T., The host response to Leishmania infection. Adv.Immunol. 2000. 74: 275–317.

4 Wilhelm, P., Ritter, U., Labbow, S., Donhauser, N., R�llinghoff, M.,Bogdan, C. and K�rner, H., Rapidly fatal leishmaniasis in resistant C57BL/6mice lacking tumor necrosis factor. J. Immunol. 2001. 166: 4012–4019.

5 Sacks, D. L. and Noben-Trauth, N., The immunology of susceptibility andresistance to Leishmania major in mice. Nat. Rev. Immunol. 2002. 2:845–858.

6 Stenger, S., Th�ring, H., R�llinghoff, M. and Bogdan, C., Tissueexpression of inducible nitric oxide synthase is closely associated withresistance to Leishmania major. J. Exp. Med. 1994. 180: 783–793.

7 Wang, Z.-E., Reiner, S. L., Zheng, S., Dalton, D. K. and Locksley, R. M.,CD4+ effector cells default to the Th2 pathway in interferon-c-deficient miceinfected with Leishmania major. J. Exp. Med. 1994. 179: 1367–1371.

8 Mattner, F., Di Padova, K. and Alber, G., Interleukin-12 is indispensable forprotective immunity against Leishmania major. Infect. Immun. 1997. 65:4378–4383.

9 Launois, P., Maillard, I., Pingel, S., Swihart, K. G., Xenarios, I., Acha-Orbea, H., Diggelmann, H. et al., IL-4 rapidly produced by Vb4Va8 CD4+

T cells instructs Th2 development and susceptibility to Leishmania major inBALB/c mice. Immunity 1997. 6: 541–549.

10 Scharton, T. M. and Scott, P., Natural killer cells are a source of IFN-c thatdrives differentiation of CD4+ T cell subsets and induces early resistance toLeishmania major in mice. J. Exp. Med. 1993. 178: 567–578.

11 Laskay, T., R�llinghoff, M. and Solbach, W., Natural killer cells participatein the early defense against Leishmania major infection in mice. Eur. J.Immunol. 1993. 23: 2237–2241.

12 Scharton-Kersten, T., Afonso, L. C. C., Wysocka, M., Trinchieri, G. andScott, P., IL-12 is required for natural killer cell activation and subsequent Thelper 1 cell development in experimental leishmaniasis. J. Immunol. 1995.154: 5320–5330.

13 Laskay, T., Diefenbach, A., R�llinghoff, M. and Solbach,W., Early parasitecontainment is decisive for resistance to Leishmania major infection. Eur. J.Immunol. 1995. 25: 2220–2227.

14 Diefenbach, A., Schindler, H., Donhauser, N., Lorenz, E., Laskay, T.,MacMicking, J., R�llinghoff, M. et al., Type 1 interferon (IFN-a/b) andtype 2 nitric oxide synthase regulate the innate immune response to aprotozoan parasite. Immunity 1998. 8: 77–87.

15 Schleicher, U., Mattner, J., Blos, M., Schindler, H., R�llinghoff, M.,Karaghiosoff, M., M�ller, M. et al., Control of Leishmania major in theabsence of Tyk2 kinase. Eur. J. Immunol. 2004. 34: 519–529.

16 Bajenoff, M., Breart, B., Huang, A. Y., Qi, H., Cazareth, J., Braud, V. M.,Germain, R. N. and Glaichenhaus, N., Natural killer cell behavior in lymphnodes revealed by static and real-time imaging. J. Exp. Med. 2006. 203:619–631.

17 de Veer, M. J., Curtis, J. M., Baldwin, T. M., DiDonato, J. A., Sexton, A.,McConville, M. J., Handman, E. and Schofield, L., MyD88 is essential forclearance of Leishmania major: possible role of lipophosphoglycan and Toll-like receptor 2 signaling. Eur. J. Immunol. 2003. 33: 2822–2831.

18 Debus, A., Gl�sner, J., R�llinghoff, M. and Gessner, A., High levels ofsusceptibility and T helper 2 response in MyD88-deficient mice infected withLeishmania major are interleukin-4 dependent. Infect. Immun. 2003. 71:7215–7218.

19 Muraille, E., De Trez, C., Brait, M., De Baetselier, P., Leo, O. and Carlier,Y., Genetically resistant mice lacking MyD88-adaptor protein display a highsusceptibility to Leishmania major infection associated with a polarized Th2response. J. Immunol. 2003. 170: 4237–4241.

20 Kropf, P., Freudenberg, M. A., Modolell, M., Price, H. P., Herath, S.,Antoniazi, S., Galanos, C. et al., Toll-like receptor 4 contributes to efficientcontrol of infection with the protzoan parasite Leishmania major. Infect.Immun. 2004. 72: 1920–1928.

21 Wagner, H., The immunobiology of the TLR9 subfamily. Trends Immunol.2004. 25: 381–386.

22 Shoda, L. K. M., Kegerreis, K. A., Suarez, C. E., Roditi, I., Corral, R. S.,Bertot, G. M., Norimine, J. and Brown, W. C., DNA from protozoanparasites Babesia bovis, Trypanosoma cruzi, T. brucei is mitogenic for Blymphocytes and stimulates macrophage expression of interleukin-12,tumor necrosis factor-a and nitric oxide. Infect. Immun. 2001. 69:2162–2171.

Eur. J. Immunol. 2007. 37: 3424–3434 Immunity to infection 3433

f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

23 Bafica, A., Scanga, C. A., Feng, C. G., Leifer, C., Cheever, A. and Sher, A.,TLR9 regulates Th1 responses and cooperates with TLR2 in mediatingoptimal resistance to Mycobacterium tuberculosis. J. Exp. Med. 2005. 202:1715–1724.

24 Drennan, M. B., Stijlemans, B., van den Abbeele, J., Quesniaux, V. J.,Barkhuizen, M., Brombacher, F., de Baetselier, P. et al., The induction of atype I immune response following a Trypanosoma brucei infection is MyD88dependent. J. Immunol. 2005. 175: 2501–2509.

25 Yasuda, K., Yu, P., Kirschning, C. J., Schlatter, B., Schmitz, F., Heit, A.,Bauer, S., Hochrein, H. and Wagner, H., Endosomal translocation ofvertebrate DNA activates dendritic cells via TLR9-dependent and -indepen-dent pathways. J. Immunol. 2005. 174: 6129–6136.

26 Zimmermann, S., Egeter, O., Hausmann, S., Lipford, G. B., R�cken, M.,Wagner, H. and Heeg, K., Cutting Edge: CpG oligodeoxynucleotides triggerprotective and curative Th1 responses in lethal murine leishmaniasis. J.Immunol. 1998. 160: 3627–3630.

27 Laouar, Y., Sutterwala, F. S., Gorelik, L. and Flavell, R. A., Transforminggrowth factor-b controls T helper type 1 cell development throughregulation of natural killer cell interferon-c. Nat. Immunol. 2005. 6:600–607.

28 Konecny, P., Stagg, A. J., Jebbari, H., English, N., Davidson, R. N. andKnight, S. C., Murine dendritic cells internalize Leishmania majorpromastigotes, produce IL-12 p40 and stimulate primary T cell proliferationin vitro. Eur. J. Immunol. 1999. 29: 1803–1811.

29 von Stebut, E., Belkaid, Y., Nguyen, B. V., Cushing, M., Sacks, D. L. andUdey, M. C., Leishmania major-infected murine Langerhans cell-likedendritic cells from susceptible mice release IL-12 after infection andvaccinate against experimental cutaneous leishmaniasis. Eur. J. Immunol.2000. 30: 3498–3506.

30 Misslitz, A. C., Bonhagen, K., Harbecke, D., Lippuner, C., Kamradt, T.and Aebischer, T., Twowaves of antigen-containing dendritic cells in vivo inexperimental Leishmania major infection. Eur. J. Immunol. 2004. 34:715–725.

31 Schleicher, U., Liese, J., Knippertz, I., Kurzmann, C., Hesse, A., Heit, A.,Fischer, J. A. et al., NK cell activation in visceral leishmaniasis requiresTLR9, myeloid DC, and IL-12, but is independent of plasmacytoid DC. J. Exp.Med. 2007. 204: 893–906.

32 Wei, X.-q., Charles, I. G., Smith, A., Ure, J., Feng, G.-j., Huang, F.-p., Xu,D. et al., Altered immune responses in mice lacking inducible nitric oxidesynthase. Nature 1995. 375: 408–411.

33 Blos, M., Schleicher, U., Rocha, F. J., Meissner, U., R�llinghoff, M. andBogdan, C., Organ-specific and stage-dependent control of Leishmaniamajor infection by inducible nitric oxide synthase and phagocyte NADPHoxidase. Eur. J. Immunol. 2003. 33: 1224–1234.

34 Huang, S., Hendriks, W., Althage, A., Hemmi, S., Bluethmann, H.,Kamijo, R., Vilcek, J. et al., Immune response in mice that lack theinterferon-c receptor. Science 1993. 259: 1742–1744.

35 Kamijo, R., Shapiro, D., Le, J., Huang, S., Aguet, M. and Vilcek, J.,Generation of nitric oxide and induction ofmajor histocompatibility complexclass II antigen in macrophages from mice lacking the interferon c receptor.Proc. Natl. Acad. Sci. USA 1993. 90: 6626–6630.

36 Himmelreich, H., Parra-Lopez, C., Tacchini-Cottier, F., Louis, J. A. andLaunois, P., The IL-4 rapidly produced in BALB/c mice after infection withLeishmania major downregulates IL-12 receptor b2 chain expression onCD4+ T cells resulting in a state of unresponsiveness to IL-12. J. Immunol.1998. 161: 6156–6163.

37 Munder, M., Eichmann, M., Moran, J. M., Centeno, F., Soler, G. andModolell, M., Th1/Th2-regulated expression of arginase isoforms in murinemacrophages and dendritic cells. J. Immunol. 1999. 163: 3771–3777.

38 Boucher, J. L., Moali, C. and Tenu, J.-P., Nitric oxide biosynthesis, nitricoxide synthase inhibitors and arginase competition for L-arginine utiliza-tion. Cell. Mol. Life Sci. 1999. 55: 1015–1028.

39 El-Gayar, S., Th�ring-Nahler, H., Pfeilschifter, J., R�llinghoff, M. andBogdan, C., Translational control of inducible nitric oxide synthase by IL-13and arginine availability in inflammatory macrophages. J. Immunol. 2003.171: 4561–4568.

40 Krug, A., French, A. R., Barchet, W., Fischer, J. A., Dzionek, A., Pingel, J.T., Orihuela, M.M. et al., TLR9-dependent recognition of MCMV by IPC and

DC generates coordinated cytokine responses that activate antiviral NK cellfunction. Immunity 2004. 21: 107–119.

41 Trinchieri, G., Interleukin-12 and the regulation of innate resistance andadaptive immunity. Nat. Rev. Immunol. 2003. 3: 133–146.

42 Moretta, A., Natural killer cells and dendritic cells: rendezvous in abusedtissues. Nat. Rev. Immunol. 2002. 2: 957–963.

43 Degli-Esposti, M. A. and Smyth, M. J., Close encounters of different kinds:dendritic cells and NK cells take centre stage. Nat. Rev. Immunol. 2005. 5:112–124.

44 Gajewski, T. F. and Fitch, F. W., Anti-proliferative effect of IFN-c in immuneregulation. I. IFN-c inhibits the proliferation of Th2 but not Th1 murinehelper T lymphocyte clones. J. Immunol. 1988. 140: 4245–4252.

45 O0Garra, A., Cytokines induce the development of functionally hetero-genous T helper cell subsets. Immunity 1998. 8: 275–283.

46 Kabelitz, D., Expression and function of Toll-like receptors inT lymphocytes.Curr. Opin. Immunol. 2007. 19: 39–45.

47 Iniesta, V., Carcelen, J., Molano, I., Peixoto, P. M. V., Redondo, E., Parra,P., Mangas, M. et al., Arginase I induction during Leishmania majorinfection mediates the development of disease. Infect. Immun. 2005. 73:6085–6090.

48 Kropf, P., Fuentes, J. M., Fahnrich, E., Arpa, L., Herath, S., Weber, V.,Soler, G. et al., Arginase and polyamine synthesis are key factors in theregulation of experimental leishmaniasis in vivo. FASEB J. 2005. 19:1000–1002.

49 Swihart, K., Fruth, U., Messmer, N., Hug, K., Behin, R., Huang, S.,Giudice, G. D. et al.,Mice from genetically resistant background lacking theinterferon-c receptor are susceptible to infection with Leishmania major butmount a polarized T helper cell 1-type CD4+ T cell response. J. Exp. Med.1995. 181: 961–971.

50 Yasuda, K., Rutz, M., Schlatter, B., Metzger, J., Luppa, P. B., Schmitz, F.,Haas, T. et al., CpG motif-independent activation of TLR9 upon endosomaltranslocation of “natural” phosphodiester DNA. Eur. J. Immunol. 2006. 36:431–436.

51 Coban, C., Ishii, K. J., Kawai, T., Hemmi, H., Sato, S., Uematsu, S.,Yamamoto, M. et al., Toll-like receptor 9 mediates innate immuneactivation by the malaria pigment hemozoin. J. Exp. Med. 2005. 201: 19–25.

52 Parroche, P., Lauw, F. N., Goutagny, N., Latz, E., Monks, B. G., Visintin,A., Halmen, K. A. et al., Malaria hemozoin is immunologically inert butradically enhances innate responses by presenting malaria DNA to Toll-likereceptor 9. Proc. Natl. Acad. Sci. USA 2007. 104: 1919–1924.

53 Bafica, A., Santiago, H. C., Goldszmid, R., Ropert, C., Gazzinelli, R. T.and Sher, A., TLR9 and TLR2 signaling together account for MyD88-dependent control of parasitemia in Trypanosoma cruzi infection. J.Immunol. 2006. 177: 3515–3519.

54 Minns, L. A., Menard, L. C., Foureau, D. M., Darche, S., Ronet, C.,Mielcarz, D. W., Buzoni-Gatel, D. and Kasper, L. H., TLR9 is required forthe gut-associated lymphoid tissue response following oral infection ofToxoplasma gondii. J. Immunol. 2006. 176: 7589–7597.

55 Mattner, F., Magram, J., Ferrante, J., Launois, P., Di Padova, K., Behin,R., Gately, M. K. et al., Genetically resistant mice lacking interleukin-12 aresusceptible to infection with Leishmania major and mount a polarized Th2cell response. Eur. J. Immunol. 1996. 26: 1553–1559.

56 Bogdan, C., Donhauser, N., D�ring, R., R�llinghoff, M., Diefenbach, A.and Rittig, M. G., Fibroblasts as host cells in latent leishmaniosis. J. Exp.Med. 2000. 191: 2121–2129.

57 Stenger, S., Donhauser, N., Th�ring, H., R�llinghoff, M. and Bogdan, C.,Reactivation of latent leishmaniasis by inhibition of inducible nitric oxidesynthase. J. Exp. Med. 1996. 183: 1501–1514.

58 Lutz, M. B., Kukutsch, N., Ogilvie, A. L., R�ssner, S., Koch, F., Romani, N.and Schuler, G., An advanced culture method for generating largequantities of highly pure dendritic cells from mouse bone marrow. J.Immunol. Methods 1999. 223: 77–92.

59 Bogdan, C., Schr�ppel, K., Lohoff, M., R�llinghoff, M. and Solbach, W.,Immunization of susceptible hosts with a soluble antigen fraction fromLeishmania major leads to aggravation of murine leishmaniasis mediated byCD4+ T cells. Eur. J. Immunol. 1990. 20: 2533–2540.

Jan Liese et al. Eur. J. Immunol. 2007. 37: 3424–34343434

f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu