The Journal of Experimental Medicine
JEM © The Rockefeller University Press $15.00
Vol. 204, No. 4, April 16, 2007 893–906 www.jem.org/cgi/doi/10.1084/jem.20061293
NK cells are key components of the innate
immune response to infectious pathogens (1, 2).
Activated NK cells are an early source of IFN-γ
and thereby contribute to the development of
type 1 Th cells (3, 4). They support the matura-
tion of DCs (5, 6) and can exhibit cytolytic
activity against host cells infected with certain
viruses, bacteria, or protozoa (7–9). The acti-
vation of NK cells is a multifactorial process
that involves soluble factors as well as stimu-
latory cell surface receptors that are triggered
during interaction with ligand-positive target
cells (10). In vitro studies revealed that human
or mouse DCs can activate resting NK cells via
direct cell–cell contact or the release of cytokines
(e.g., IFN-α/β, IL-2, IL-12, and IL-18;
reference 6). Depletion of a subset of lymphoid
DCs (CD8α+) by anti-CD8 antibody treatment,
which is not selective for DCs, partially abro-
gated fms-like tyrosine kinase 3 ligand (Flt3L)-
induced and NK cell–mediated tumor regression
and impaired the expansion of Ly49H+ NK
cells during murine cytomegalovirus infection
(11, 12), but in vivo evidence for DC-dependent
regulation of NK cell IFN-γ expression and
cytotoxicity has not yet been published.
More recent in vitro studies on human DC
populations and NK cells suggested that in ad-
dition to conventional or myeloid DCs (mDCs;
CD11chigh) plasmacytoid DCs (pDCs) can also
activate NK cells for cytolytic activity in a type
I IFN-α/β–dependent manner (13, 14). Mouse
pDCs express a unique selection of myeloid
and lymphoid cell surface markers (CD11b−,
NK cell activation in visceral leishmaniasis
requires TLR9, myeloid DCs, and IL-12,
but is independent of plasmacytoid DCs
Ulrike Schleicher,1,2 Jan Liese,1 Ilka Knippertz,2 Claudia Kurzmann,1
Andrea Hesse,1,2 Antje Heit,3 Jens A.A. Fischer,4 Siegfried Weiss,5
Ulrich Kalinke,6 Stefanie Kunz,1 and Christian Bogdan1,2
1Institute of Medical Microbiology and Hygiene, University of Freiburg, D-79104 Freiburg, Germany
2Institute of Clinical Microbiology, Immunology and Hygiene, University of Erlangen, 91054 Erlangen, Germany
3Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, 81675 München, Germany
4Department of Research and Development, Miltenyi Biotec GmbH, 51429 Bergisch-Gladbach, Germany
5Department of Molecular Immunology, Helmholtz Zentrum für Infektionsforschung, D-38124 Braunschweig, Germany
6Department of Immunology, Paul Ehrlich Institute, 63225 Langen, Germany
Natural killer (NK) cells are sentinel components of the innate response to pathogens, but
the cell types, pathogen recognition receptors, and cytokines required for their activation
in vivo are poorly defi ned. Here, we investigated the role of plasmacytoid dendritic cells
(pDCs), myeloid DCs (mDCs), Toll-like receptors (TLRs), and of NK cell stimulatory cytokines
for the induction of an NK cell response to the protozoan parasite Leishmania infantum.
In vitro, pDCs did not endocytose Leishmania promastigotes but nevertheless released
interferon (IFN)-훂/훃 and interleukin (IL)-12 in a TLR9-dependent manner. mDCs rapidly
internalized Leishmania and, in the presence of TLR9, produced IL-12, but not IFN-훂/훃.
Depletion of pDCs did not impair the activation of NK cells in L. infantum–infected mice.
In contrast, L. infantum–induced NK cell cytotoxicity and IFN-후 production were abolished
in mDC-depleted mice. The same phenotype was observed in TLR9−/− mice, which lacked
IL-12 expression by mDCs, and in IL-12−/− mice, whereas IFN-훂/훃 receptor−/− mice showed
only a minor reduction of NK cell IFN-후 expression. This study provides the fi rst direct
evidence that mDCs are essential for eliciting NK cell cytotoxicity and IFN-후 release in vivo
and demonstrates that TLR9, mDCs, and IL-12 are functionally linked to the activation of
NK cells in visceral leishmaniasis.
Abbreviations used: BM-mDC,
BM-derived myeloid DC;
BM-MΦ, BM-derived macro-
phages; BM-pDC, BM-derived
plasmacytoid DC; DT, diphthe-
ria toxin; DTR, DT receptor;
Flt3L, fms-like tyrosine kinase 3
ligand; gDNA, genomic DNA;
GU, guanosine-uridine; IRF,
IFN regulatory factor; IFNAR,
IFN-α/β receptor; kDNA,
kinetoplast DNA; mDC,
myeloid DC; MM, metallophilic
macrophage; MOI, multiplicity
of infection; MyD88, myeloid
diff erentiation factor 88; MZM,
marginal zone macrophage;
pDC, plasmacytoid DC;
ssRNA, single-stranded RNA;
TLR, Toll-like receptor.
I. Knippertz and A. Hesse’s present address is University
Clinic for Dermatology, University of Erlangen, D-91052
The online version of this article contains supplemental material.
PDCS, MDCS, TLR9, AND LEISHMANIA | Schleicher et al.
CD11cint, B220+, Ly6C+, Gr-1+, CD62L+, and CD45RA+;
reference 15). They release large amounts of IFN-α/β in vitro
and in vivo in response to DNA or RNA viruses, bacterial
DNA, or synthetic oligodeoxynucleotides (ODNs) with un-
methylated CpG motifs (CpG ODN; reference 15), synthetic
guanosine-uridine (GU)-rich single-stranded RNA (ssRNA;
references 16 and 17), or purifi ed or synthetic hemozoin (18).
In each of these cases, the production of IFN-α/β was de-
pendent on Toll-like receptor (TLR)7 or TLR9, which are
typically expressed by mouse pDCs (19). Both TLRs signal
through the adaptor molecule myeloid diff erentiation factor
88 (MyD88), which recruits further signaling molecules and
fi nally leads to the activation of NF-κB or IFN regulatory
factor (IRF)-7, followed by rapid IFN-α/β expression (20).
A protective immune response against intracellular proto-
zoan parasites of the genus Leishmania is characterized by the
induction and expansion of IFN-γ–producing CD4+ and
CD8+ T cells, which activate macrophages for the expression
of antileishmanial eff ector pathways such as inducible nitric
oxide synthase (21, 22). In the mouse models of experimental
cutaneous (e.g., Leishmania major) and visceral (e.g., Leishmania
donovani and Leishmania infantum) leishmaniasis, NK cells were
found to participate in the innate immune response and con-
trol of the parasites (23–31). Endogenous and exogenous
IL-12 or IFN-α/β were shown to confer NK cell activation
(25, 28–30, 32, 33) and/or protective immunity in these
models (21, 25, 29, 34–37). Furthermore, in vitro stimulation
assays with pro- or amastigote parasites and certain mouse
DCs (38–40), ex vivo immunohistochemical or fl ow cyto-
metry analyses of lymphatic tissues (41, 42), as well as DC trans-
fer and vaccination studies (43) identifi ed DCs as a source of
IL-12 during Leishmania infections. However, it is unknown
how Leishmania parasites are initially sensed by the immune
system to trigger an innate NK cell response during the early
phase of Leishmania infection and whether the activation of
NK cells in vivo requires interaction with CD11chigh mDCs,
CD11cint pDCs, and/or mDC-/pDC-derived cytokines.
In this study, we investigated which DC population,
pathogen recognition receptor, and cytokine is essential for
the induction of NK cell cytotoxicity and IFN-γ production
in visceral leishmaniasis. Our parallel analysis of pDCs and
mDCs revealed that only pDCs, but not mDCs, produced
IFN-α/β after stimulation with L. infantum promastigotes,
whereas pDCs as well as mDCs released IL-12. The Leishmania-
induced production of both cytokines was strictly depen-
dent on TLR9. Unexpectedly, pDCs (and, to a large extent,
IFN-α/β receptor [IFNAR] signaling) were dispensable for
the innate NK cell response to L. infantum in vivo. Instead,
the activation of splenic NK cells after L. infantum infection
required CD11chigh mDCs, TLR9, and IL-12.
Differential production of IFN-훂/훃 and IL-12p40 by pDCs
and mDCs in response to Leishmania promastigotes
Although mDCs are well-known targets of Leishmania, the
interaction of pDCs with Leishmania has not yet been studied.
Therefore, we investigated whether pDCs are targets of
Leishmania parasites. Flt3L-expanded BM cells of C57BL/6
mice were exposed to Leishmania promastigotes and ana-
lyzed for the expression of costimulatory surface mole-
cules. L. infantum and L. major promastigotes up-regulated
the expression of CD40, CD80, and CD86 on mPDCA-
1+CD11b−CD11c+ BM-derived pDCs (BM-pDCs; as well
as on CD11b+CD11c+ BM-derived mDCs [BM-mDCs])
within the Flt3L culture, although to a lesser extent than
the known pDC activator CpG ODN 2216 (Fig. S1 A,
which is available at http://www.jem.org/cgi/content/full/
jem.20061293/DC1, and not depicted; reference 19).
Figure 1. IFN-훂/훃 expression in pDCs versus mDCs. Cells were stim-
ulated with 1 μM CpG ODN 2216, 50 ng/ml poly(I:C), 200 ng/ml LPS,
L. infantum, L. major, or L. braziliensis promastigotes (MOI = 3) ± anti-
mCD40 mAb (5 μg/ml). (A) IFN-α/β production of sorted C57BL/6
BM-pDCs (Flt3L-BM culture) or BM-mDCs (GM-CSF-BM culture) after
stimulation for 48 h. Mean ± SEM of two experiments. (B) IFN-α and
IFN-β mRNA expression of sorted C57BL/6 BM-pDCs after stimulation for
24 h as determined by real-time RT-PCR. Mean (±SD) of the calculated
relative expression of seven independent experiments. (C) IFN-α/β pro-
duction of purifi ed splenic pDCs or splenic mDCs of 129Sv mice stimu-
lated in parallel. Mean ± SEM of two experiments. ▼, not detectable.
JEM VOL. 204, April 16, 2007
Next, we compared the cytokine expression of C57BL/6
BM-pDCs (CD11b−CD11c+CD62L+ cells sorted from
Flt3L-expanded BM cultures) and BM-mDCs (CD11b+-
CD11c+CD86low cells sorted from GM-CSF– expanded
BM cultures) after stimulation with live Leishmania promasti-
gotes (multiplicity of infection [MOI] = 3, unless stated
otherwise). pDCs generated copious amounts of IFN-α/β
as detected by bioassay (Fig. 1 A) or ELISA (Fig. S1 C).
Among all Leishmania species tested (L. major, L. infantum,
and Leishmania braziliensis), L. infantum induced the highest
release of IFN-α/β protein in 48 of a total of 58 experi-
ments, which frequently was only one order of magnitude
lower than the amount of IFN-α/β elicited by HSV-1 virus
or CpG ODN 2216 (Fig. 1 A and Fig. S1 B). Real-time
RT-PCR revealed a 10–1,000-fold induction of the mRNA
expression of IFN-β and, with the exception of IFN-α11
and IFN-α13, of all IFN-α subtypes tested (Fig. 1 B). mDCs,
in contrast, produced strikingly less IFN-α/β in response to
CpG ODN 2216 and virtually no IFN-α/β after exposure
to Leishmania parasites (Fig. 1 A).
Leishmania promastigotes also activated purifi ed splenic
pDCs (CD11b−CD11cintGr-1+) for the production of IFN-
α/β, whereas no IFN-α/β was detectable in the culture
supernatants of purifi ed splenic mDCs (CD11chighMHCII+;
Fig. 1 C). Poly(I:C), which targets TLR3, and LPS, which
interacts with TLR4, were inactive on BM-pDCs and splenic
pDCs but elicited an IFN-α/β response in BM-mDCs and
splenic mDCs (Fig. 1, A and C, and not depicted).
BM-pDCs co-cultured with Leishmania promastigotes
released TNF, but not MIP-2α or nitric oxide (not depicted).
Both BM-pDCs and BM-mDCs produced IL-12p40 after
stimulation with CpG ODN 2216 or Leishmania promasti-
gotes. The CpG- or Leishmania-induced IL-12p40 release of
mDCs was higher compared with pDCs and enhanced by
anti-CD40 (see WT pDCs and mDCs in Fig. 2 D). IL-12p70
remained undetectable (not depicted).
These data demonstrate that Leishmania promastigotes
potently activate mouse pDCs, but not mDCs, for the ex-
pression of IFN-α/β mRNA and protein, whereas both DC
populations are triggered for the release of IL-12p40.
The induction of IFN-훂/훃 and/or IL-12 in pDCs and mDCs
by Leishmania requires TLR9
The data presented above raised the question of which patho-
gen recognition receptor(s) on the surface of pDCs and
mDCs transmits the signal for the induction of IFN-α/β
and/or IL-12 by Leishmania.
Mouse pDCs express a very limited spectrum of TLRs
(TLR7 and TLR9), which all signal via the MyD88 adaptor
molecule (19). When sorted WT and MyD88−/− BM-pDCs
were exposed to Leishmania promastigotes or CpG ODN
2216, we found that both the CpG- and the Leishmania-
induced IFN-α/β release were entirely dependent on MyD88
(Fig. 2 A). The IFN-α/β production in response to L. infantum
or L. major was completely abolished in the absence of TLR9
in seven out of eight experiments (in one experiment, the
IFN-α/β release was ?100 U/ml). After stimulation of
TLR9−/− BM-pDCs with L. braziliensis, IFN-α/β was un-
detectable in three of eight experiments and reduced by >95%
in the remaining experiments (Fig. 2 B). As expected, CpG
ODN 2216 did not induce any IFN-α/β in TLR9−/− pDCs
(Fig. 2 B), whereas synthetic GU-rich ssRNA, a TLR7 ligand
(16, 17), clearly triggered the release of IFN-α/β (mean U/ml ±
SEM of two experiments: 8,789 ± 3,096).
The production of IFN-α/β during viral infections or in
response to certain TLR9 ligands is regulated by a positive
feedback loop in which the early secreted IFN-β and IFN-α4
initiate further IFN-α/β expression via IFNAR-mediated de
novo synthesis of IRF-7 (15, 20). As pDCs constitutively ex-
press high levels of IRF-7 and release huge amounts of IFN-α
even in the absence of an autocrine feedback loop (15, 44), we
tested whether the Leishmania-induced IFN-α/β production
Figure 2. Receptors involved in Leishmania-induced expression of
IFN-훂/훃 and/or IL-12p40. Sorted BM-pDCs (from Flt3L-BM culture)
and sorted immature BM-mDCs (from GM-CSF-BM culture) of C57BL/6
WT, MyD88−/−, TLR9−/−, IFN-β−/−, or IFNAR−/− mice were analyzed.
After stimulation with 1 μM CpG ODN 2216, L. infantum, L. major, or
L. braziliensis promastigotes (MOI = 3) for 48 h, the (A–C) IFN-α/β
content (VSV bioassay) or the (D) IL-12p40 content (ELISA) of the
respective culture supernatants was determined. Mean ± SEM of three
(A and C), eight (B), or two independent experiments (D). ▼, not detect-
able. Signifi cant differences between WT and KO cells are indicated as
follows: *, P < 0.05; **, P < 0.01; ***, P < 0.005.
PDCS, MDCS, TLR9, AND LEISHMANIA | Schleicher et al.
requires endogenous IFN-α/β signaling. No IFN-α/β was
detectable in the cultures of sorted C57BL/6 BM-pDCs defi -
cient for the IFNAR chain 1 (IFNAR−/−) that were stimu-
lated with Leishmania parasites or CpG ODN 2216. The
IFN-α/β production triggered by Leishmania was also mark-
edly reduced in IFN-β−/− pDCs (Fig. 2 C).
In both BM-pDCs and BM-mDCs, the IL-12p40 pro-
duction elicited by the TLR9 ligand CpG ODN 2216 or by
viable Leishmania promastigotes was entirely dependent on
the presence of TLR9 (Fig. 2 D).
Collectively, these data illustrate that TLR9 is essential for
the Leishmania-mediated induction of two major NK cell–
activating cytokines (IFN-α/β and IL-12) in pDCs and
mDCs. In addition, the generation of IFN-α/β by pDCs co-
cultured with Leishmania promastigotes requires an IFNAR-
dependent feedback loop that is partially maintained by IFN-β.
Leishmania-induced IFN-훂/훃 production by pDCs does not
require replication, stage maturation, viability, or uptake
of the parasites and can be mimicked by Leishmania DNA
To characterize the parasite requirements for the induction
of IFN-α/β, we exposed sorted C57BL/6 BM-pDCs to
(a) viable promastigotes of the logarithmic or stationary growth
phase; (b) viable, but irradiated (i.e., replication-defi cient)
promastigotes; (c) freeze-thaw lysates of stationary-phase
Leishmania promastigotes; or to (d) boiled lysates of Leishmania
promastigotes. In all these cases, the induction of IFN-α/β
was in the same order of magnitude (Fig. 3 A, top, and not
depicted), indicating that neither a specifi c parasite stage nor
a productive infection of the host cells is required and that
proteins are unlikely to be the only active component of
Leishmania promastigotes. However, when pDCs were cul-
tured with viable L. infantum promastigotes in the presence of
DNase, we observed a signifi cant (approximately fourfold)
reduction of the IFN-α/β content in the culture superna-
tants (Fig. 3 A, top). This raised the possibility that Leishmania
DNA at least partially accounts for the stimulatory activity of
Eukaryotic DNA contains unmethylated CpG-DNA
motifs, which might cause TLR9-dependent stimulation of
immune cells (45–47). When C57BL/6 BM-pDCs were
stimulated with 0.5–5 μg/ml genomic DNA (gDNA) from
L. infantum promastigotes, the production of IFN-α/β was
similar to that after co-culture with viable L. infantum para-
sites at an MOI of 3 (Fig. 3 A, bottom). At low concentra-
tions (0.1 μg/ml), L. infantum gDNA induced only small
(<300 U/ml; six experiments) or undetectable quantities of
IFN-α/β (eight experiments; Fig. 3 A, bottom), whereas the
equivalent number of whole parasites (MOI = 1.2, i.e., cor-
responding to 0.1 ug/ml L. infantum gDNA) still potently
induced IFN-α/β (Fig. S1 B).
No IFN-α/β was measurable when TLR9−/− pDCs
were stimulated with L. infantum gDNA or when WT pDCs
were exposed to L. infantum lysates treated with DNase,
L. infantum gDNA treated with DNase, L. infantum mito-
chondrial kinetoplast DNA (kDNA), mouse splenic gDNA,
human blood gDNA, or to apoptotic (UV-irradiated or
peroxynitrite-treated) annexin V+ pDCs. The DNase used did
not exert nonspecifi c inhibitory eff ects because the IFN-α/β–
inducing activity of ssRNA remained unaltered (Fig. 3 A, top
and bottom, and not depicted). Thus, the TLR9-dependent
IFN-α/β release is parasite gDNA specifi c and is not due to
the recognition of self-DNA.
As TLR9 is expressed in endosomal and lysosomal com-
partments and requires an acidic pH to interact with ODN or
DNA ligands (48), we tested whether chloroquine, which pre-
vents endosomal acidifi cation, can inhibit Leishmania-induced
IFN-α/β release. Both the CpG ODN– and the Leishmania-
induced IFN-α/β production were blocked by chloroquine
without detectable toxicity as assessed by trypanblue exclu-
sion (not depicted). This fi nding suggested that Leishmania
Figure 3. Parasite requirements for TLR9-dependent Leishmania-
specifi c IFN-훂/훃 production of pDCs. (A) IFN-α/β activity (VSV bio-
assay) in the culture supernatants of FACS-sorted C57BL/6 or TLR9−/−
BM-pDCs stimulated for 48 h with viable L. infantum promastigotes
(MOI = 3) in the absence or presence of 500 U/ml DNase I, L. infantum
freeze-thaw lysate (MOI = 3, without or with DNase I treatment), 5 μg/ml
of synthetic GU-rich ssRNA (without or with DNase I treatment), or
500 U/ml DNase I alone (A, top; mean ± SEM of three experiments) or with
L. infantum promastigotes (MOI = 3), L. infantum gDNA, or L. infantum
kDNA (A, bottom; mean ± SEM of two experiments). Signifi cant differ-
ences (WT vs. KO, gDNA vs. kDNA) are indicated as follows: *, P < 0.05;
**, P < 0.01; ***, P < 0.005. (B) C57BL/6 BM-MΦ, sorted BM-mDCs, or
sorted C57BL/6 BM-pDCs (from Flt3L-BM culture) were stimulated with
L. infantum promastigotes (MOI = 3) for 16 h. After staining of the cell
surface (anti-CD11c or anti–Siglec-H) and of the parasites, the number
of infected cells, noninfected cells, or cells with attached parasites was
determined microscopically by evaluation of at least 100 cells in different
visual fi elds. Mean ± SEM of six (pDCs), four (mDCs), or three (BM-MΦ)
separate experiments. Examples of the double immunofl uorescence
staining of pDCs or mDCs and L. infantum are shown in the lower panels.
▼, not detectable. Bar, 10 μM.
JEM VOL. 204, April 16, 2007
promastigotes might enter an acidic endosomal compartment
of pDCs. However, multicolor fl uorescent microscopy of
sorted C57BL/6 pDCs (CD11b−CD11c+CD62L+), mDCs
(CD11b+CD11c+), and BM-derived macrophages (BM-MΦ)
revealed that mDCs and BM-MΦ, but not pDCs, internalize
L. infantum promastigotes. Importantly, parasites were attached
to the surface of ?10% of the pDCs (Fig. 3 B). When parasites
and pDCs were separated by a transwell membrane, no induc-
tion of IFN-α/β was observed (64,049 and 64,618 U/ml vs.
<1 U/ml in two experiments).
Collectively, these data indicate that the activation of
pDCs by Leishmania promastigotes for the production of IFN-
α/β requires cell–cell contact, but not uptake of the parasites.
Leishmania gDNA is a potent inducer of IFN-α/β and is likely
to contribute to the stimulatory activity of intact parasites.
The NK cell response to L. infantum in vivo
is weakly impaired in IFNAR−/− mice and unaffected
in pDC-depleted mice
The strong activation of pDCs for the release of IFN-α/β by
Leishmania promastigotes in vitro led us to investigate whether
this process also occurs in vivo and is relevant for the initia-
tion of the NK cell response to the parasite. We used a model
of visceral (i.e., hepatic, splenic, and BM) leishmaniasis (49)
because BM-derived and splenic pDCs were highly respon-
sive to our viscerotropic strain of L. infantum and therefore
might also sense the parasite in vivo.
In C57BL/6 WT mice, i.v. infection of L. infantum led to
a striking induction of NK cell cytotoxic activity that was
maintained in IFNAR−/− mice (Fig. 4 A). In the spleen,
L. infantum caused an increased IFN-γ mRNA expression
within 8 h of infection (Fig. S2, available at http://www
.jem.org/cgi/content/full/jem.20061293/DC1) and the pro-
duction of IFN-γ protein by NK cells. The percentage of
IFN-γ+ cells within the NK1.1+CD3− splenic NK cell pop-
ulation was similar in WT and IFNAR−/− mice at the 12-h
time point but was signifi cantly reduced at 24 h of infection
in the IFNAR−/− group (Fig. 4 B). These results suggest that
the L. infantum–induced and IFNAR-dependent secretion of
IFN-α/β by pDCs that we observed in vitro is only partially
involved in the activation of NK cells and/or does not occur
to the same extent in vivo. In line with the latter possibility,
we consistently found only a very weak induction (approxi-
mately factor 3–4) of IFN-α/β mRNAs in L. infantum–
infected C57BL/6 WT mice (Fig. S2; see also WT mice in
Fig. S4 A and Fig. 8 C), which was comparable in IFNAR−/−
mice (Fig. S2). In addition, IFN-α or IFN-β protein was not
detectable by ELISA in the serum or plasma of various strains
of WT mice (C57BL/6, 129Sv, and BALB/c) within 4–24 h
after L. infantum infection (not depicted).
To directly address the role of pDCs, we injected the
pDC-specifi c anti–PDCA-1 mAb twice before infection,
which led to an ?80–90% reduction of the Siglec-
H+CD11c+CD11b− splenic pDCs at all time points of infec-
tion tested (not depicted). This depletion protocol completely
blocked the pDC-dependent IFN-α production in mice (50)
elicited by i.v. injection of CpG ODN (Fig. S3, available at
In contrast, control IgG–treated or pDC-depleted C57BL/6
or 129Sv mice infected with L. infantum showed comparable
levels of NK cell cytotoxicity, IFN-γ production, and splenic
IFN-α4 and IFN-β mRNA expression (Fig. 4, C and D, and
not depicted). Thus, pDCs are unlikely to control the early
NK cell response to L. infantum.
The innate NK cell response to L. infantum requires IL-12
and CD11chigh DCs
Because IFN-α/β had only a limited impact on the NK cell
activation during the early phase of L. infantum infection, we
tested whether IL-12 controls the onset of the NK cell re-
sponse in visceral leishmaniasis. i.v. infection with L. infantum
led to strong NK cell cytotoxicity and IFN-γ expression in
WT mice, but not in IL-12p35/p40−/− (devoid of IL-12 and
IL-23) or IL-12p35−/− mice (devoid of IL-12; Fig. 5, A–D).
Thus, IL-12 is essential for the activation of NK cells in
L. infantum–infected mice. Given that mDCs were more potent
producers of IL-12 than pDCs (notably after cross-linking of
CD40; Fig. 2 D) and that the depletion of pDCs did not
prevent the L. infantum–induced NK cell response (Fig. 4,
C and D), we postulated that the IL-12–dependent NK cell
activation in vivo is driven by mDCs.
Figure 4. Role of IFN-훂/훃 and pDCs for NK cell cytotoxicity and
IFN-후 expression in the spleens of mice infected i.v. with 107
L. infantum promastigotes. (A and B) C57BL/6 WT versus IFNAR−/− mice.
Mean ± SEM of four experiments with one to two mice per time point and
mouse group. (C and D) Splenic pDCs of C57BL/6 mice were depleted by
injection of 500 μg anti–mPDCA-1 mAb 24 and 4 h before infection with
L. infantum. Control mice received rat IgG. Mean ± SEM of three experiments
(C) or one of three experiments (D). (A and C) 24 h after injection of PBS or
L. infantum, spleen cells were prepared and NK cell cytotoxic activity was
measured. Infected WT mice were signifi cantly different from PBS controls
(*, P < 0.05; **, P < 0.01), but not from IFNAR−/− or anti–mPDCA-1–treated
mice. (B and D) 12 and 24 h after infection, spleen cells were restimulated
in medium ± YAC tumor cells (ratio 1:1) and stained for CD3−NK1.1+ NK
cells and intracellular IFN-γ. **, P < 0.01 WT versus IFNAR−/−.
PDCS, MDCS, TLR9, AND LEISHMANIA | Schleicher et al.
To investigate this directly, we used CD11c-diphtheria
toxin (DT) receptor (DTR)/GFP mice that carry a transgene
encoding a fusion protein of DTR and GFP under control of
the promoter of the mouse CD11c gene (51). 2 d after injec-
tion of DT, CD11chighMHCII+CD11b+ splenic mDCs were
ablated, whereas pDCs and all other CD11cint cells remained
unaff ected (Fig. 6 A). In agreement with a previous report
(52), we observed that the DT treatment not only depleted
CD11chighMHCII+ mDCs, but also MOMA-1+ metallo-
philic macrophages (MMs) and ERTR-9+ marginal zone
macrophages (MZMs) in the spleen (Fig. 6 B). However, 5 d
after DT treatment, CD11chighMHCII+ mDCs had repopu-
lated the spleen to a large, albeit varying extent in individu-
ally analyzed mice (Fig. 6 C), whereas ERTR-9+ MZMs
remained completely depleted, and only very few MOMA-1+
MMs became visible in all 10 individually analyzed mice
(Fig. 6 D). F4/80+ red pulp macrophages were not signifi -
cantly aff ected by the DT treatment in CD11c-DTR/GFP
mice (51, 52; not depicted), and the percentage of CD11b+
cells even increased at days 2 and 5 after DT treatment (Fig.
6 A and not depicted).
When WT and CD11c-DTR/GFP mice were treated
with DT, infected with L. infantum 24 h later, and analyzed
12 or 24 h after infection (i.e., at day 2 after DT), up-regula-
tion of IFN-γ mRNA in the spleen, activation of NK cells,
and IL-12p40 production of mDCs were seen in infected
WT mice, but not in the transgenic mice (Fig. 7, A and B,
and Fig. S4, A and B, which is available at http://www.jem
the number of splenic NK cells (CD3−NK1.1+) and their
expression of IFN-γ after in vitro activation with the DC-
independent stimuli PMA/ionomycin was comparable in
DT-treated naive or infected WT and CD11c-DTR/GFP
mice, except for a more prominent IFN-γ production in the
case of infected WT mice, which refl ects the prior DC-
dependent priming of NK cells by L. infantum in vivo (Fig.
S5, A and B). The L. infantum–induced up-regulation of
IFN-α4, IFN-α5, and IFN-β mRNA at day 2 after DT was
?1,000-fold higher in the spleen of CD11chigh mDC-
depleted mice than in the respective control mice (Fig. S4 A),
which, however, did not rescue the NK cell response in those
mice. At day 5 after DT, in contrast, NK cell cytotoxicity and
IFN-γ production after L. infantum infection were clearly
restored in CD11c-DTR/GFP mice (Fig. 7, C and D).
Considering the varying degree of restoration of NK cell
activation, the spleens of 10 CD11c-DTR/GFP mice were
split and analyzed in parallel by FACS, immunohistology,
Figure 5. NK cell cytotoxicity and IFN-후 expression in the spleens
of C57BL/6 WT versus IL-12p35/p40−/− and BALB/c WT versus
IL-12p35−/− mice infected i.v. with 107 L. infantum promastigotes.
(A and C) 24 h after injection of PBS or L. infantum, spleen cells were
prepared and NK cell cytoxic activity was measured. Mean ± SEM of two
experiments (two mice/group). (B and D) 12 and 24 h after infection,
spleen cells of WT and KO mice were restimulated in medium ± YAC
tumor cells (ratio 1:1) and stained for CD3−NK1.1+ NK cells and intra-
cellular IFN-γ. One (B) or mean ± SEM (D) of two experiments with one
to two mice per time point and mouse group. (A, C, and D) The values
obtained for infected WT mice are signifi cantly different from WT PBS con-
trols and from infected KO mice. *, P < 0.05; **, P < 0.01; ***, P < 0.005.
Figure 6. Phenotypic analysis of the spleens of C57BL/6 WT and
CD11c-DTR/GFP transgenic mice 2 or 5 d after i.p. injection of DT
and 12–24 h after i.v. infection with 107 L. infantum promastigotes.
(A and C) Flow cytometric analysis of DC populations in the spleen. The
percentage of the respective cell population is given in the plot panels.
(B and D) Immunohistological staining of MZMs (ERTR-9+) and MMs
(MOMA-1+) in the spleen. Nuclei were counterstained with Meyer’s hemalaun.
Bar, 100 μm. (A and B) One of fi ve experiments, with two to three mice
per mouse group. (C and D) One of two experiments, with 2–10 mice
per mouse group. In C, two individual mice of the group of a total of 10
infected CD11c-DTR/GFP mice with different DC reconstitution are shown.
JEM VOL. 204, April 16, 2007
and NK cell cytotoxicity assays, which revealed that the mag-
nitude of NK cell cytotoxicity solely correlated with the per-
centage of reconstitution of CD11chighMHCII+ mDCs (r2 =
0.77; Fig. S6). MZMs and MMs were uniformly absent and
the distribution and expression level of CD11b+ or F4/80+
in the red pulp were comparable in all mice tested (not de-
picted). A similar correlation was seen between NK cell IFN-γ
production and the reconstitution of CD11chighMHCII+
mDCs (r2 = 0.66; not depicted). We conclude that activation
of NK cells during the innate phase of L. infantum infection
requires IL-12 and the presence of CD11chigh DCs, but not
of MZMs or MMs.
TLR9 is required for the production of IL-12 and the
activation of NK cells in L. infantum–infected mice
The critical role of mDCs and IL-12 for the activation of NK
cells in L. infantum–infected mice and the TLR9-dependent
production of IL-12 by mDCs in vitro led us to investigate
whether TLR9 is essential for the expression of IL-12 and the
initiation of the NK cell response in vivo. As observed
before, i.v. infection of WT mice with L. infantum promasti-
gotes was followed by a rapid induction of NK cell cytotoxicity
and IFN-γ protein expression in the spleen. In TLR9−/−
mice, in contrast, no NK cell activation was measurable in
response to L. infantum (Fig. 8, A and B). Both the number
of NK cells as well as the activation of NK cells by the TLR3
ligand poly(I:C) remained unaltered, indicating that the dele-
tion of TLR9 does not lead to the depletion or to a universal
functional suppression of NK cells (Fig. 8 B and not depicted).
The diff erence in the IFN-γ expression between WT and
TLR9−/− mice during the innate phase of L. infantum infec-
tion was also seen by quantitative RT-PCR analysis of RNA
samples prepared from total splenic tissue. In accordance with
the TLR9-dependent production of type I IFNs and IL-12p40
by pDCs and/or mDCs in vitro, the L. infantum– induced ex-
pression of IFN-α4, IFN-β, and IL-12p40 mRNA was re-
duced in TLR9−/− compared with WT mice. However, due
to the cell type–restricted expression and up-regulation
of type I IFNs and IL-12, the factors by which the mRNA
levels of these cytokines were increased in the spleen of WT
mice after infection were not high enough to allow for sig-
nifi cant diff erences between WT and TLR9−/− mice for all
genes, at all time points of infection, and in each individual
experiment (Fig. 8 C and not depicted). To defi nitively
answer the question of TLR9-dependent expression of IL-12
in vivo, we resorted to single cell analyses using intracellular
cytokine staining. In the spleen of L. infantum–infected mice,
IL-12p40 protein was exclusively found in CD11chigh mDCs
(?1% of all living spleen cells; see Fig. 6 A), but not in
F4/80+ macrophages (not depicted). As shown in Fig.
8 D, the L. infantum–induced production of IL-12p40 by
CD11chigh mDCs in the spleen and its accumulation in the
plasma of WT mice were absent in TLR9−/− mice. IL-12p70
was not detectable in the plasma samples (not depicted).
Collectively, these data show that TLR9 is strictly re-
quired for the activation of NK cells in visceral leishmaniasis
because it is essential for the production of IL-12 by CD-
In the past, only few studies had analyzed the role of NK cells
in visceral leishmaniasis. In untreated Indian patients with vis-
ceral leishmaniasis, the cytolytic activity of peripheral blood
NK cells was reduced (53). In experimental visceral leishman-
iasis, NK cell–defi cient beige mice (bg/bg) failed to eliminate
L. donovani (23). After transfer of syngeneic cloned NK cells
into bg/bg mice, the splenic parasite burdens were indistin-
guishable from those of normal WT mice (24). Finally, a 7-d
treatment of BALB/c mice with IL-12 led to a 70% reduction
of the liver parasite load compared with untreated control
mice, whereas in IL-12–treated but NK cell–depleted mice,
the decrease of the parasite numbers was only 30% (25). Col-
lectively, all these earlier observations argued for a protective
function of NK cells during primary visceral leishmaniasis.
Figure 7. NK cell cytotoxicity and IFN-후 expression in C57BL/6
WT and CD11c-DTR/GFP transgenic mice 2 or 5 d after i.p. injection
of DT and 12–24 h after i.v. infection with 107 L. infantum pro-
mastigotes or injection of PBS. (A) NK cell cytotoxicity of splenocytes
at day 2 after DT. Mean ± SEM of fi ve experiments, with two to three
mice per time point and mouse group. ***, P < 0.005, WT L. infantum
compared with WT PBS and CD11c-DTR L. infantum. (B) Intracellular IFN-γ
staining of NK cells (CD3−NK1.1+) in splenocytes at day 2 after DT that
were restimulated in medium ± YAC tumor cells (ratio 1:1). Staining of
individual mice of one experiment is shown (day 5 analysis of the same
experiment is illustrated in D). Similar results were obtained in fi ve inde-
pendent experiments. (C) NK cell cytotoxicity of splenocytes at day 5 after
DT. Results of one of two similar experiments are shown, with 2–10 mice
per mouse group. *, P < 0.05; **, P < 0.01, WT and CD11c-DTR L. infantum–
infected mice compared with the respective PBS control. (D) Intracellular
IFN-γ staining of NK cells in splenocytes at day 5 after DT (restimulated
as in B). One of two independent experiments.
PDCS, MDCS, TLR9, AND LEISHMANIA | Schleicher et al.
However, neither the process of innate NK cell activation
nor the underlying mechanisms have been investigated in
To the best of our knowledge, this study is the fi rst to dem-
onstrate that the activation of NK cell functions in vivo
requires the presence of mDCs (but not pDCs). Previous
analyses in tumor or viral models were based on the applica-
tion of anti-CD8 antibodies (which deplete CD8α+ lym-
phoid cells without being selective for these cells) and/or did
not directly investigate the eff ector functions of NK cells
(cytotoxicity and IFN-γ production; references 11, 12, and 54).
Another novelty of our report is the strict requirement of
TLR9 for the induction of an NK cell response to a nonviral
pathogen. This is particularly surprising, because unlike DNA
viruses that were shown to drive NK cell activation in a
TLR9-dependent manner (55, 56), Leishmania parasites are
complex, eukaryotic pathogens that express a wide variety of
possible ligands for pattern recognition receptors. Our re-
sults show that the early NK cell response to a visceral infec-
tion with L. infantum is completely dependent on TLR9,
CD11chigh mDCs, and IL-12, weakly impaired in the ab-
sence of IFN-α/β, and unaff ected after depletion of pDCs.
Therefore, this study off ers a coherent picture of the receptor,
cellular, and cytokine requirements of NK cell activation
in vivo. Finally, the essential role of TLR9 for innate NK cell
activation observed here might serve as a plausible explana-
tion for the previously reported TLR9 dependency of adap-
tive Th1 immune responses in other microbial infection
models (45, 46, 57).
pDCs, IFN-훂/훃, and TLR-9–dependent NK cell activation
Exposure of pDCs to promastigotes of diff erent Leishmania
species led to a rapid induction of IFN-β and a broad spec-
trum of IFN-α isoforms. The IFN-α/β release was not de-
pendent on the parasite stage, replication, viability, integrity,
or uptake but required a direct contact between the pDCs
and the promastigotes, and an acidifi cation of endosomal/
lysosomal compartments that are thought to carry the TLR9
receptor (19, 48). Genomic, but not the evolutionary ancient
mitochondrial (kinetoplast) DNA of Leishmania parasites, was
suffi cient to mimic the TLR9-dependent induction of IFN-
α/β by intact promastigotes. At fi rst glance, the TLR9-
dependent and chloroquin-inhibitable stimulation of pDCs
by intact promastigotes is diffi cult to reconcile with the
Figure 8. NK cell cytotoxicity, NK cell IFN-후 production, and
cytokine expression in the spleens of WT and TLR9−/− mice in-
fected i.v. with 107 L. infantum promastigotes. (A) NK cell cytoxicity
of splenocytes at 24 h after infection. Mean ± SEM of four independent
experiments. *, P < 0.05, WT L. infantum compared with WT PBS or
TLR9−/− L. infantum. (B) 12 h after injection of L. infantum, PBS or poly
(I:C) (50 μg, i.p.) spleen cells of WT and TLR9−/− mice were restimulated
in medium ± YAC tumor cells (ratio 1:1) and stained for NK cells
(CD3−NK1.1+) and intracellular IFN-γ. One of three similar experiments.
(C) IFN-α4, IFN-β, IFN-γ, IL-12p35, and IL12p40 mRNA expression in the
spleens 3, 6, 9, 12, and 24 h after infection. PBS control mice were set as
1 (mean ± SEM of three experiments; in each experiment two mice per
time point and mouse group were analyzed by real-time RT-PCR with
triplicate determinations for each gene). Inset: Relative cytokine mRNA
expression levels (compared with the mHPRT-1 housekeeping gene) in
the spleen 6 h after infection (mean ± SEM of three experiments).
(D) Top panels: 12 h after injection of PBS or L. infantum, spleen cells of
WT and TLR9−/− mice were restimulated in medium and stained for CD11c+
DCs and intracellular IL-12p40 protein (one of three similar experiments;
the percentage of IL-12p40+ cells is indicated in the panels; mean ±
SEM of three independent experiments is shown in the graph below).
***, P < 0.001, WT L. infantum compared with WT PBS and TLR9−/−
L. infantum. Bottom: IL-12p40 plasma levels of 12-h–infected or PBS
control mice as measured by ELISA (mean ± SEM of three independent
experiments). ***, P < 0.001, WT L. infantum compared with WT PBS and
TLR9−/− L. infantum.
JEM VOL. 204, April 16, 2007
absence of detectable parasite uptake in pDC/Leishmania co-
cultures. However, as extrusion of gDNA by viable eukaroytic
cells has been observed (58), and DNase treatment signifi -
cantly (?75%) reduced the production of IFN-α/β by pDCs
exposed to Leishmania, our results are compatible with the
idea that gDNA released by promastigotes during their at-
tachment to pDCs accounts for the strong induction of IFN-
α/β. We wish to point out that our fi ndings do not exclude
the possibility that another, so far unknown, heat-resistant
component of Leishmania functions as TLR9 ligand and con-
tributes to the activation of pDCs to produce IFN-α/β by
intact parasites. In this respect, it is noteworthy that at least
one non-DNA ligand of TLR9, i.e., hemozoin (a degradation
product of host cell–derived heme in Plasmodium-infected
erythrocytes), has been described that triggered human, but not
mouse, pDCs for the release of IFN-α/β (18, 59).
In light of the strong production of IFN-α/β by pDCs
exposed to L. infantum in vitro, the observation that a 90%
reduction of the number of pDCs in the spleen did not aff ect
the Leishmania-induced NK cell response was unexpected. As
there was only a limited up-regulation of type I IFN mRNAs
in the infected spleen that was not altered by the depletion of
pDCs, splenic pDCs might not be targeted and/or activated
by L. infantum in vivo during the early phase of infection. In
this respect, visceral leishmaniasis diff ers from viral infection
models in which pDC depletion resulted in a decrease of the
expression of IFN-α (55, 60). However, even in viral infec-
tions pDCs are not necessarily required for NK cell cytotoxic
activity (55). The fact that pDCs were dispensable for the
elicitation of an innate NK cell response in L. infantum–
infected mice does not exclude that the interaction between
pDCs and Leishmania might be relevant during later stages of
Our analysis of IFNAR−/− mice revealed that IFN-α/β
(possibly released by cells other than pDCs) contributes to the
induction of NK cell IFN-γ expression, but not of NK cell
cytotoxicity in L. infantum–infected mice. This contrasts with
(a) the role of IFN-α/β in mouse cytomegalovirus–infected
mice, in which IFN-α/β was required for the induction of
NK cell cytotoxicity, but not IFN-γ expression (61); and (b)
with the function of IFN-α/β in experimental cutaneous
leishmaniasis, where anti–IFN-α/β drastically reduced both
the NK cell cytotoxicity and the early IFN-γ peak in the
draining lymph nodes of L. major–infected mice (28). pDCs
have been detected in the lymph nodes of L. major–infected
mice (62), but whether they are necessary for the innate NK
cell response and account for the previously reported early
IFN-α/β production in cutaneous leishmaniasis (28) has not
yet been investigated.
CD11chigh mDCs, IL-12, and TLR9-dependent
NK cell activation
CD11c-DTR/GFP transgenic mice (51) are currently the
only mouse model to eliminate CD11chigh DCs. To the best
of our knowledge, this is the fi rst study in which these mice
have been used to demonstrate that CD11chigh DCs are
necessary for the priming of NK cell functions (IFN-γ pro-
duction and cytotoxicity) in vivo. Recently, it was reported
that DT treatment of CD11c-DTR/GFP mice, in addition
to CD11chigh DCs, also depletes MZMs and MMs in the
spleen (52), which we could confi rm in both naive and
L. infantum–infected mice (Fig. 6 and not depicted). Although
viscerotropic Leishmania parasites were previously shown to
selectively activate DCs, but not MZMs, of the spleen (41),
we carefully addressed the potential role of MZMs and MMs
for the activation of NK cells. The time course and functional
analyses shown in Fig. 6, Fig. 7, and Fig. S6 demonstrated
that in L. infantum–infected, DT-treated CD11c-DTR/GFP
mice, NK cell activation was rapidly restored once CD11chigh
DCs repopulated the spleen, although at these time points
both MZMs and MMs were still absent. Thus, NK cell acti-
vation requires CD11chigh DCs, whereas MZMs and MMs
are clearly dispensable. It is also unlikely that the absence of
NK cell activation in L. infantum–infected, DT-treated
CD11c-DTR/GFP mice results from a suppressive or toxic
eff ect of DT or dead DCs on NK cells. First, DT treatment
did not reduce the number of NK cells. Second, DT did not
impair the response of NK cells to PMA/ionomycin
(Fig. S5). Third, the function of NK cells was fully restored
after the repopulation of CD11chigh DCs, which further
excludes a cytotoxic or long-lasting suppressive eff ect of DT
on splenic NK cells.
The TLR9-dependent induction of IL-12p40 by L. infantum
in mDCs in vitro (Fig. 2 D) and in vivo (Fig. 8 D), the equally
absent innate NK cell response to L. infantum in TLR9−/−,
IL-12−/−, and CD11chigh-depleted mice (Figs. 5, 7, and 8),
and the lack of infection-induced IL-12p40 expres sion in
CD11chigh-depleted mice (Fig. S4 B) strongly argues for the
following model of NK cell activation in which TLR9,
mDCs, IL-12, and NK cells are coherently linked: L. infantum
activates CD11chigh DCs via TLR9 for the generation of
IL-12 that subsequently triggers NK cell cytotoxicity and
IFN-γ production. The dominant role of mDCs and IL-12 is
further underlined by the observation that the depletion of
CD11chigh mDCs before L. infantum infection did not diminish
the number of splenic pDCs (Fig. 6 A) and was accompanied
by a roughly 1,000-fold increase of the IFN-α and IFN-β
mRNA expression (Fig. S4 A), which, however, was insuffi -
cient to prevent the loss of NK cell activity. Our results also
off er a mechanistic and functional explanation for the close
and prolonged interactions between NK cells and mDCs that
were recently seen in the lymph nodes of mice infected with
L. major (experimental cutaneous leishmaniasis) using intra-
vital microscopy (31).
Human NK cells were previously shown to express
TLRs, including TLR2, TLR3, and TLR9 (63–65). This led
us to consider whether L. infantum might directly activate
mouse NK cells in a TLR9-dependent manner. Previous
and present observations strongly argue against this possi-
bility. First, FACS-sorted, IL-2–expanded mouse splenic
NK1.1+CD3− NK cells did not express TLR9 mRNA as
assessed by RT-PCR (unpublished data). Second, FACS-sorted,
PDCS, MDCS, TLR9, AND LEISHMANIA | Schleicher et al.
IL-2–expanded or MACS-sorted (DX5+) naive splenic NK
cells were neither activated by CpG ODN (55) nor by Leish-
mania promastigotes (unpublished data) for the expression
of IFN-γ or cytolytic activity. Third, in the absence of
CD11chigh DCs or IL-12, no NK cell activity was detectable
in L. infantum–infected mice.
In summary, this study revealed an unexpected, dominant
role of TLR9 for the initiation of the NK cell response to a
complex eukaryotic pathogen. Although initial in vitro ex-
periments suggested that both pDCs and mDCs are valid
candidates to deliver activating signals to NK cells, our in
vivo analyses demonstrated that TLR9, CD11chigh mDCs,
and IL-12, but not pDCs and type I IFNs, were essential for
NK cell cytotoxicity and IFN-γ production in visceral leish-
maniasis. The observed TLR9- and IL-12–dependent NK
cell activation by mDCs suggests a certain hierarchy within
the receptors, cell types, and cytokines previously shown to
exert activating eff ects on NK cells, which might also hold
true for infections with other intracellular pathogens.
MATERIALS AND METHODS
Mice, parasites, and infection
Female C57BL/6, BALB/c, and 129Sv (PasIco) mice were from Charles River
Laboratories, and CD11c-DTR/GFP transgenic mice (15th generation back-
cross to C57BL/6; reference 51) were from The Jackson Laboratory. Breeding
pairs of IL-12p35−/− mice (fi fth generation backcross to BALB/c; reference 34)
and IL-12p35/p40−/− mice (10th generation backcross to C57BL/6; ref-
erence 34) were provided by G. Alber (University of Leipzig, Leipzig, Germany)
and H. Mossmann (Max Planck Institute for Immunobiology, Freiburg,
Germany), respectively. IFNAR−/− (66), IFN-β−/− (67), MyD88−/− (68),
and TLR9−/− mice (69) were backcrossed to C57BL/6 mice for 10, 13, 8, or 10
generations, respectively. All mice were housed under specifi c pathogen-free
conditions and used at the age of 6–12 wk.
Promastigotes of L. major (MHOM/IL/81/FEBNI; reference 70),
L. infantum (MHOM/00/98/LUB1; reference 71), and L. braziliensis (MHOM/
BR/94/H-3227; reference 72) were grown from amastigotes isolated from
skin lesions of BALB/c (L. major and L. infantum) or iNOS−/− (L. braziliensis)
mice and propagated in vitro (70).
For infection, mice were injected i.v. in the retro-orbital vein or in the
tail vein with 300 μl PBS or 107 stationary phase L. infantum promastigotes
in 300 μl PBS. The animal experiments were approved by the animal wel-
fare committee of the Regierungspräsidium Freiburg.
In vivo treatment
To deplete pDCs, mice were i.p. injected with 500 μg rat anti–mPDCA-1
mAb or control rat IgG (The Jackson Laboratory) at 24 and 4 h before i.v.
injection of L. infantum, 5 μg of a phosphorothioate-modifi ed CpG ODN
(50), or PBS. The CpG ODN was mixed with 30 μl of the cationic lipo-
some preparation DOTAP (Roche Diagnostics) in a volume of 300 μL PBS
(50). 4, 6, 8, 12, and 24 h after infection, spleen cells were analyzed for the
presence of CD11b−CD11cintSiglec-H+ cells to control depletion. To de-
plete mDCs, CD11c-DTR/GFP mice received an i.p. injection of DT
(4 ng/g body weight; Sigma-Aldrich) 1 or 4 d before L. infantum infection.
The reduction of CD11chighMHCII+ mDCs was controlled by FACS analy-
sis. The depletion of macrophages in DT-treated CD11c-DTR/GFP mice
was monitored by immunohistology of the spleen. For activation of NK cells
in vivo, mice received i.p. 50 μg poly(I:C) (Sigma-Aldrich).
For surface phenotyping and cell sorting, the following unconjugated,
fl uorochrome (FITC-, PE-, or APC-) -labeled or biotinylated mAbs
were used (all from BD Biosciences unless otherwise stated): anti-CD11b
(M1/70), anti-Ly6C (ER-MP20; BMA Biomedicals), anti-Ly6G (Gr-1),
anti-CD62L (MEL-14), anti-CD11c (HL3), anti-CD45R/B220 (RA3-
6B2), anti–I-A/I-E (M5/114.15.2), anti-CD40 (3/23), anti-CD80 (16-
10A1), anti-CD86 (GL1), anti-NK1.1 (PK136), anti-CD49b (DX5),
anti-CD3 (145-2C11), anti–mPDCA-1(Miltenyi Biotec), and anti–Siglec-H
(440c; Hycult Biotechnology). Biotinylated antibodies were detected by
streptavidin-APC or streptavidin-PE (BD Biosciences). The specifi city of
the stainings was verifi ed by the use of isotype control mAbs. Propidium
iodide was included at 1 μg/ml in the fi nal wash to detect dead cells. All
analyses were performed on a FACSCalibur (BD Biosciences) applying the
CELLQuest Pro software. The FL3 channel was used to exclude propidium
iodide+ dead cells.
Generation and purifi cation of pDCs and mDCs
Splenic pDCs and mDCs. After collagenase D (Roche Diagnostics) treat-
ment, splenic DCs were enriched from total spleen cells by positive selection
of CD11c+ cells using anti-CD11c MicroBeads and AutoMACS (Miltenyi
Biotec). pDCs were obtained by MoFlo sorting (Cytomation Inc.) of
CD11b−CD11cintGr-1+ cells, and mDCs were obtained by sorting of
CD11b+CD11chigh cells (purity ≥95%). Purifi ed mDCs also expressed
MHC class II.
BM-pDCs. BM-pDCs were generated from total BM cells in the presence
of Flt3L (73). After incubation in red blood cell lysis buff er (Sigma-Aldrich),
BM cells were cultured in complete RPMI with 100 ng/ml rmFlt3L (R&D
Systems) for 7–8 d at 2 × 106 cells/ml (25 cm2 cell culture fl asks, 5 ml).
At day 4, 2.5 ml of medium per flask was replaced by 2.5 ml of fresh
medium with 50 ng/ml Flt3L. After 7–8 d, 75–95% of the cells were
CD11c+. pDCs (CD11b−) represented 57–77% and mDCs (CD11b+) rep-
resented 23–43% of the CD11c+ population (unpublished data). At day 7 or 8,
CD11b−CD11c+CD62L+ BM-pDCs were purifi ed by MoFlo sorting
(purity >95%). The purifi ed pDCs were readily stained with anti-B220 and
the pDC-specifi c mAbs anti–Siglec-H (74) and anti–mPDCA-1 (15 and
BM-mDCs. BM-mDCs were either sorted as CD11c+CD11b+ cells from
Flt3L-expanded BM-cultures (see above) or generated from BM cells incubated
with rmGM-CSF (75). GM-CSF–expanded BM cultures (day 8) contained
?80% CD11b+CD11chigh mDCs, which were further purifi ed as immature
CD11b+CD11c+CD86low mDCs by MoFlo sorting (purity ≥96%).
Stimulation of pDCs and mDCs
pDCs and mDCs were cultured in 96-well (105 cells/well, 250 μL), 24-well
(106 cells/well, 1 ml), or 24-transwell tissue culture plates (106 cells/well;
0.4-μm pore size, 700 μL; Corning Costar) at 37°C and 5% CO2/95%
humidifi ed air using RPMI 1640 (Invitrogen) that was supplemented with
50 μM 2-ME, 1% nonessential amino acids, 1 mM sodium pyruvate, 100 μg/ml
kanamycin sulfate, and 10% FCS (PAA Laboratories) for pDC cultures or
supplemented as described previously (29) plus 10% FCS (PAA) for mDC
and all other cell cultures. Cells were activated for 12–72 h with 1 μM CpG
ODN 2216 (Thermo Electron), 200 ng/ml LPS (Escherichia coli O111:B4;
Sigma-Aldrich), 50 ng/ml poly(I:C) (Sigma-Aldrich), 20 ng/ml rmIFN-γ
(provided by G. Adolf, Boehringer Ingelheim, Vienna, Austria), 5 μg/ml
anti-mCD40 (clone 3/23; BD Biosciences), UV-inactivated HSV-1 (pro-
vided by T. Stamminger, University of Erlangen, Erlangen, Germany),
Leishmania spp. promastigotes (logarithmic stage or stationary growth phase;
parasite/pDC ratio [MOI] = 3:1, unless indicated diff erently), 160 mJ/cm2
of UV-irradiated L. major or L. infantum promastigotes (MOI = 3:1), Leish-
mania spp. antigen (freeze-thaw lysates of promastigotes; MOI = 3:1), 0.1–5
μg/ml gDNA or kDNA of L. infantum promastigotes or with GU-rich
ssRNA double-right complexed with LyoVec (5μg/ml; ssRNA-DR/
LyoVec; InvivoGen). In some experiments, ssRNA, Leishmania DNA, or
Leishmania lysates were digested with 500 U/ml of bovine pancreas DNase I
(Sigma-Aldrich) before their addition to the pDC cultures according to the
manufacturer’s protocol. gDNA of Leishmania spp. was prepared by proteinase
JEM VOL. 204, April 16, 2007
K digestion of promastigotes, followed by phenol/chloroform extraction
and ethanol precipitation or by using the Blood&Cell Culture DNA kit
(QIAGEN). In both cases, RNA was removed with DNase-free RNaseA
(Invitrogen). kDNA was prepared as described previously (76) and con-
trolled by gel electrophoresis.
Cytokine and nitrite measurements
IFN-α/β levels were determined with a L929/vesicular stomatitis virus
protection assay using triplicates and serial twofold dilutions of the culture
supernatants (28). Purifi ed mouse IFN-α/β and a neutralizing sheep anti–
IFN-α/β antiserum (provided by I. Gresser, Institute Curie, Paris, France)
was used as a standard or to ascertain that all antiviral activity in the
supernatants was due to IFN-α/β. The content of IFN-α (including IFN-α1,
IFN-α4, IFN-α5, IFN-α6, and IFN-α9) or IFN-β (both from PBL
Biomedical Laboratories), TNF (sensitivity 40 pg/ml; R&D Systems), MIP-2
(sensitivity 20 pg/ml; Nordic Biosite), IL-12p40, or IL-12p70 (sensitivity
40 pg/ml; BD Biosciences) was measured by ELISA. NO2
by the Griess assay (77).
− was determined
Intracellular cytokine staining
IFN-γ staining in NK cells. Spleen cells of infected or control mice were
restimulated for 6 h in the presence of 10 μg/ml brefeldin A with medium
alone or with YAC-1 tumor target cells (ratio 1:1) for repeated priming of
the NK cells or with 50 ng/ml PMA (Sigma-Aldrich) and 750 ng/ml iono-
mycin (Sigma- Aldrich). After staining of NK cell surface markers (CD3-
NK1.1+ or CD3-DX5+), the cells were fi xed with CytopermCytofi x (BD
Biosciences) for 20 min and incubated with APC-conjugated rat anti–mouse
IFN-γ (XMG1.2; BD Biosciences) as described previously (77).
IL-12p40 staining in CD11c+ cells. Applying the same method as
described above, spleen cells were restimulated with medium alone or, as a
positive control, with 1 μM CpG ODN 1668 (Thermo Electron). For sur-
face staining, anti-CD11c, anti-CD11b, and anti-F4/80 (Cl:A3-1; Serotec)
mAbs were used. IL-12p40 was stained by an APC-conjugated rat anti–
mouse IL-12p40/p70 mAb (C15.6; BD Biosciences).
NK cell cytotoxicity
After determining the percentage of NK1.1+CD3− or DX5+CD3− NK
cells within whole spleen cells, the NK cell cytotoxicity against YAC-1
tumor cells was determined (27, 28).
Immunofl uorescence microscopy
Purifi ed BM-pDCs and BM-mDCs or BM-MΦ (77) were incubated with
Leishmania promastigotes for 16 h. Thereafter, nonadherent pDCs and mDCs
were transferred to adhesion slides (Marienfeld Lab. Glassware), whereas
macrophages were directly stained in LabTek Permanox Chambers (Nalge
Nunc Int.). For double immunofl uorescence staining, the cells were blocked
with PBS/1% BSA and incubated with biotinylated anti-CD11c (HL3;
mDCs), anti–Siglec-H (pDCs), or anti-CD11b (macrophages) antibodies
(BD Biosciences), followed by Cy3-conjugated streptavidin (Invitrogen).
Internalized Leishmania parasites were stained after fi xation in 3% parafor-
maldehyde and permeabilization in 1% saponin using human anti–L. major
antiserum (78) and fl uorescein–labeled anti–human IgG (Fab′)2 fragments
(Dianova). Vectashield (containing DAPI to stain the nuclei; Vector Labora-
tories) -mounted slides were analyzed with an ApoTome-equipped Axio-
plan2 microscope connected to an AxioCam-MR digital camera (Carl Zeiss
Immunohistochemistry of splenic acetone-fi xed cryosections (5–6 μm) were
performed using unconjugated rat anti-F4/80 (CI:A3-1), rat anti–mouse
macrophage (MOMA-1), and rat anti–SIGN-R1 (ERTR-9) mAbs (all from
BMA Biomedicals), followed by biotin-conjugated (Fab′)2 fragments of
mouse anti–rat IgG or goat anti–rat IgM (μ chain specifi c; both from
Dianova), alkaline phosphatase–conjugated streptavidin (DakoCytomation),
and by a red alkaline phosphatase substrate (Vector Laboratories). Sections
were counterstained with Meyer’s hemalaun, mounted with Aquatex
(Merck), and analyzed by light microscopy (Axioskop 2 plus; Carl Zeiss
RNA preparation and PCR
Total RNA was prepared using the RNeasy extraction kit (QIAGEN).
Contaminant gDNA was removed with DNase (DNAfree; Ambion). The
presence of gDNA was excluded by performing a PCR reaction with
1 μL of the RNA sample as template and primers for mouse β-actin (sense:
5′-C A C C C G C C A C C A G T T C G C C A -3′; antisense: 5′-C A G G T C C C-
G G C C A G C C A G G T -3′). Total RNA (1–10 μg) was reverse transcribed
using the High Capacity cDNA Archive kit (Applied Biosystems). Sub-
sequent real-time PCR was performed on an ABI Prism 7900 sequence
detector (Applied Biosystems) using Taqman Universal Mastermix and
Assays-on-Demand (Applied Biosystems), which include forward and reverse
primers and the FAM-labeled probe for the target gene, respectively. The
following assays were used: murine hypoxanthine guanine phosphoribosyl
transferase 1 (mHPRT-1; Mm00446968_m1), mIFN-α2 (Mm00833961_s1),
mIFN-α4 (Mm00833969_s1), mIFN-α5 (Mm00833976_s1), mIFN-α6
(Mm01258374_s1), mIFN-α9 (Mm00833983_s1), mIFN-α11 (Mm01257312_
s1), mIFN-α12 (Mm00616656_s1), mIFN-α13 (Mm00781548_s1), mIFN-α14
(Mm01703465_s1), mIFN-β (Mm00439546_s1), mIL-12p35 (Mm00434165_
m1), mIL-12p40 (Mm00434170_m1), mIFN-γ (Mm00801778_m1), and
TLR9 (Mm00446193_m1). Each cDNA was amplifi ed and measured in
triplets with 50–100 ng cDNA per well in a reaction volume of 15 μL and
the following cycle conditions: 2 min at 50°C, 10 min at 95°C, and then 15 s
at 95°C and 60 s at 60°C for 40 cycles. mRNA levels were calculated with
the SDS 2.1 software (Applied Biosystems). The amount of mRNA of each
gene was normalized to the housekeeping gene mHPRT-1. mRNA expres-
sion levels were calculated as the n-fold diff erence relative to the housekeep-
ing gene by the formula: relative expression = 2−(CT(target)−CT(mHPRT-1)).
Statistical analysis was performed using the unpaired Student’s t test.
Online supplemental material
Fig. S1 shows the up-regulation of costimulatory molecules and the dose-
dependent induction of IFN-α/β (determined by bioassay or ELISA) in
pDCs upon exposure to Leishmania promastigotes. Fig. S2 presents quantita-
tive RT-PCR data on the expression of IFN-α/β and IFN-γ in C57BL/6
WT and IFNAR−/− mice infected with 107 L. infantum. Fig. S3 illustrates
the reduction of IFN-α production in mice challenged with CpG after prior
depletion of pDCs. Fig. S4 shows the splenic expression of IFN-γ mRNA
and type I IFN mRNAs as well as the expression of IL-12p40 protein in
splenic DCs in DT-treated C57BL/6 WT and CD11c-DTR/GFP trans-
genic mice. Fig. S5 illustrates the percentage of NK cells and the PMA/ion-
omycin-induced expression of IFN-γ protein by NK cells in the spleens of
C57BL/6 WT and CD11c-DTR/GFP transgenic mice after injection of
DT and infection with L. infantum. Fig. S6 documents the linear correlation
between NK cell cytotoxicity and DC reconstitution in the spleens of
C57BL/6 WT and CD11c-DTR/GFP transgenic mice. Figs. S1–S6 are
available at http://www.jem.org/cgi/content/full/jem.20061293/DC1.
We are grateful to G. Alber (University of Leipzig, Germany), S. Bauer (University
of Marburg, Germany), D. Busch (University of Munich, Germany), M. Colonna
(Washington University, St. Louis), I. Gresser (Institut Curie, Paris), A. Krug
(University of Munich, Germany), T. Stamminger (University of Erlangen,
Germany), and P. Aichele and A. Diefenbach (Institute of Medical Microbiology
and Hygiene, Freiburg) for advice or the generous supply of mice, reagents,
This work was supported by the priority program “Innate Immunity” of the
German Research Foundation (Bo 996/3-1, 3-2, 3-3), by the Collaborative Research
Center 620 “Immunodefi ciency” of the German Research Foundation (project A9),
and by the European Community (QLK2-CT-2001-02103).
The authors have no confl icting fi nancial interests.
PDCS, MDCS, TLR9, AND LEISHMANIA | Schleicher et al.
Submitted: 19 June 2006
Accepted: 2 March 2007
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