Download Lack of CXCR3 Delays the Development of Hepatic Inflammation

BRIEF REPORT
Lack of CXCR3 Delays
the Development of Hepatic
Inflammation but Does Not Impair
Resistance to Leishmania donovani
Joseph Barbi,1 Steve Oghumu,1 Lucia E. Rosas,1 Tracy Carlson,1
Bao Lu,2 Craig Gerard,2 Claudio M. Lezama-Davila,1
and Abhay R. Satoskar1
Department of Microbiology, The Ohio State University, Columbus; 2Ina Sue
Perlmutter Laboratory, Children’s Hospital, Harvard Medical School, Boston,
Massachusetts
1
CXC chemokine receptor 3 (CXCR3) ligands CXCL9 and
CXCL10 are produced at high levels in mice and humans
infected with Leishmania donovani, but their contribution
to host resistance against L. donovani is not clear. Here, using
CXCR3⫺/⫺ mice, we demonstrate that, although CXCR3 regulates early immune cell trafficking and hepatic inflammation during L. donovani infection, it is not essential for immunity against L. donovani, unlike L. major. CXCR3⫺/⫺
C57BL/6 mice show a delayed onset of hepatic inflammation
and granuloma formation after L. donovani infection. However, they mount an efficient T helper cell type 1 response,
recruit T cells to the liver, and control parasite growth as
efficiently as do CXCR3+/+ C57BL/6 mice.
Leishmania donovani is an intracellular protozoan parasite that
causes visceral leishmaniasis (VL). VL is characterized by dissemination of parasites into the spleen, liver, lymph nodes, and
bone marrow, resulting in hepatosplenomegaly, fever, abdominal pain, and weight loss. The disease is fatal if left untreated,
because of complications such as secondary bacterial infection,
anemia, and malnutrition [1].
CXCR3 is expressed on a variety of leukocytes, including
CD4+ Th1 cells. Three of the main ligands for CXCR3 are
Received 28 September 2006; accepted 21 November 2006; electronically published 16
April 2007.
Potential conflicts of interest: none reported.
Presented in part: ASTMH, 55th Annual Meeting, Atlanta, November 2006; Woods Hole
Immunoparasitology Meeting, Woods Hole, April 2007.
Financial support: National Institutes of Health (grant AI51328).
Reprints or correspondence: Dr. Abhay R. Satoskar, Dept. of Microbiology, The Ohio State
University, 484 W. 12th Ave., Columbus, OH 43221 ([email protected]).
The Journal of Infectious Diseases 2007; 195:1713–7
2007 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2007/19511-0021$15.00
DOI: 10.1086/516787
CXCL9 (also known as “monokine induced by interferon
[IFN]–g,” or MIG), CXCL10 (also known as “IFN-inducible
protein 10,” or IP-10), and CXCL11 (also known as “IFNinducible T cell a-chemoattractant,” or I-TAC), and their induction is closely associated with Th1-dominated responses [2].
CXCR3 and its ligands play a critical role in the recruitment
of leukocytes to inflammatory sites in several diverse models
of disease [3]. Additionally, some experiments have shown that
CXCR3 ligands also regulate T cell activation and IFN-g production [4].
Experiments conducted in our laboratory have shown that
CXCR3 is required for the resolution of primary L. major infection [5]. In the absence of CXCR3, L. major–resistant C57BL/
6 mice mount a Th1 response but recruit fewer CD4+ and CD8+
T cells to infected skin, produce less IFN-g locally, and develop
lesions full of parasites [5]. In contrast, wild-type mice effectively control parasite growth and show a significant increase
in CXCR3-expressing T cells in both the regional lymph nodes
and the lesions [5]. These findings indicate that CXCR3 plays
a nonredundant role in the trafficking of IFN-g–producing
effector T cells to infected skin during cutaneous leishmaniasis.
CXCR3 and its ligands also regulate recruitment of T cells
to the liver during inflammation [6]. Furthermore, patients
with VL show elevated levels of CXCL9 and CXCL10 in their
serum during active infection [7]. Despite these observations,
it is not clear whether CXCR3 and its ligands contribute to
host resistance against L. donovani. Therefore, we examined the
in vivo role played by CXCR3 in mediating host resistance
against VL caused by L. donovani by use of CXCR3⫺/⫺ C57BL/
6 mice. Our results demonstrate that, although CXCR3 may
contribute to the development of hepatic inflammation initially,
it is not essential for Th1 development, leukocyte recruitment,
and resolution of L. donovani infection.
Materials and methods. Eight- to 10-week-old, sexmatched CXCR3⫺/⫺ C57BL/6 and wild-type C57BL/6 mice
(Harlan) were infected intravenously with 1 ⫻ 107 L. donovani
(1 Sudan strain) amastigotes harvested from the spleens of
infected hamsters. Mice were housed and maintained in conformity with the institutional guidelines for animal research at
the Ohio State University. Disease progression was monitored
by measuring parasite loads in livers and spleens 15, 30, and
60 days after infection, as described elsewhere [8]. Also, hematoxylin-eosin–stained tissue sections from the livers were
examined at these time points, to enumerate granulomas as
described elsewhere [8]. Furthermore, spleens were removed
from these mice, and parasite-specific T cell proliferation was
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assessed by Alamar Blue assay (Biosource International). Levels
of IFN-g, interleukin (IL)–12p70, IL-4, and IL-10 in supernatants were measured by ELISA, as described elsewhere [8].
For flow cytometry analysis, single-cell suspensions of liver
leukocytes were obtained as described elsewhere [8]. Cells
(1 ⫻ 10 6–2 ⫻ 10 6) were stained with fluorescein isothiocyanate–
and phycoerythrin-conjugated antibodies purchased from Biolegend and BD PharMingen. Single-cell suspensions of splenocytes were obtained as described for the proliferation assay
and were stained in a manner similar to the liver cells. Flow
cytometry analysis was then performed using a FACSCalibur
flow cytometer and CellQuest Pro software (Becton Dickinson).
Total RNA was extracted from infected livers by use of the
SV Total RNA Isolation System (Promega). mRNA was reverse
transcribed, and cDNA was amplified in an Opticon2 DNA
Engine (BioRad) using SYBR Green Taq polymerase. The primers used to amplify the cDNA in this semiquantitative reversetranscription polymerase chain reaction (RT-PCR) were found
using the PrimerBank Web site (http://pga.mgh.harvard.edu/
primerbank/index.html). In all of the experiments, Student’s
unpaired t test was used to determine the statistical significance
of differences in the values observed. P ! .05 was considered to
be significant.
Results and discussion. We have previously demonstrated
that CXCR3 is required for immunity against cutaneous leishmaniasis caused by L. major [5]. In the present study, however,
CXCR3⫺/⫺ C57BL/6 mice did not show increased susceptibility
to L. donovani and did control L. donovani growth in the liver
and spleen as efficiently as resistant wild-type C57BL/6 (figure
1A and 1B). Interestingly, 30 days after infection, CXCR3⫺/⫺
mice showed more-rapid resolution of parasite burden in their
spleens than did CXCR3+/+ mice, indicating that a lack of
CXCR3 facilitates parasite clearance from the spleen (figure 1B).
Together, these findings demonstrate that CXCR3 is not required for the control of L. donovani infection in mice. Furthermore, they also suggest that a CXCR3-dependent mechanism may be involved in promoting L. donovani growth in the
spleen.
CXCR3 controls the migration of CD4+ and CD8+ T cells to
the site of inflammation in several diseases [3, 9], and CXCR3⫺/⫺
Figure 1. Course of Leishmania donovani infection in CXCR3+/+ and CXCR3⫺/⫺ C57BL/6 mice. Liver (A) and spleen (B) parasite loads were determined
15, 30, and 60 days after intravenous inoculation of 1 ⫻ 107 L. donovani amastigotes. Parasite burdens in the liver and spleen are expressed as
mean SE Leishman-Donovan units (LDU). At these time points, hematoxylin-eosin–stained liver sections were also examined to assess inflammation
and enumerate granulomas (C). Granuloma counts represent the mean SE nos. of granulomas per 10 high-power fields (hpf) for 5 mice/group at
each time point. Panel D shows histological analysis of infected livers from CXCR3+/+ and CXCR3⫺/⫺ mice. Parasite loads are mean values for 12
mice/group at each time point, obtained in 3 independent experiments with similar results. *P ! .05, unpaired Student’s t test.
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Figure 2. Analysis of cytokine responses in the spleens and livers of Leishmania donovani–infected CXCR3+/+ and CXCR3⫺/⫺ C57BL/6 mice. Spleen
cells from infected mice were stimulated during a 72-h incubation with 20 mg/mL L. donovani antigen, and interleukin (IL)–12p70 (A), interferon (IFN)–
g (B), IL-10 (C), and IL-4 (D) production was measured by ELISA. mRNA levels of IL-12 (E), IFN-g (F), tumor necrosis factor (TNF)–a (G), IL-10 (H), and
IL-4 (I) in infected livers were measured by real-time reverse-transcription polymerase chain reaction. Results were normalized to the housekeeping
gene GAPDH and are presented as fold induction relative to that in uninfected CXCR3⫺/⫺ or CXCR3+/+ mice. Results for all experiments are the
mean SE values for triplicate samples from 11–12 mice/group at each time point, obtained in 3 independent experiments. *P ! .05.
C57BL/6 mice show a significant impairment of T cell recruitment to the skin during L. major infection [5]. Effector CD4+
and CD8+ T cells recruited to the liver during L. donovani
infection are critical for the host defense against L. donovani,
but they also contribute to granuloma formation and induce
liver disease. We therefore analyzed hepatic inflammation and
enumerated granulomas in the livers of L. donovani–infected
CXCR3+/+ and CXCR3⫺/⫺ mice on days 15, 30, and 60 after
infection. The onset of hepatic inflammation and granuloma
formation was delayed in CXCR3⫺/⫺ mice, and these mice had
significantly fewer foci of inflammation and granulomas in their
livers than did CXCR3+/+ mice on days 15 and 30 after infection
(figure 1C and 1D). However, both groups eventually developed
similar hepatic inflammation and contained comparable numbers of granulomas by day 60 (figure 1C and 1D). Flow cytometry analysis showed that the infected livers of CXCR3⫺/⫺
mice contained 50% less CD11b+ macrophages, CD4+ T cells,
and CD8+ T cells on day 15 than did CXCR3+/+ mice, but both
groups contained comparable numbers of these cell types in
their livers at later time points (data not shown). These findings
indicate that, despite a delayed onset of cellular infiltration and
inflammation, CXCR3⫺/⫺ mice ultimately recruit sufficient effector immune cells to the site of L. donovani infection. Our
observations are similar to those of a previous study, which
reported that a lack of CXCR3 impairs early granuloma formation during Mycobacterium tuberculosis infection in mice but
has no effect on the final outcome of the infection; furthermore,
this study also found that neutrophils control granuloma formation via a CXCR3-dependent pathway [10]. However, it is
unlikely that a lack of neutrophil recruitment to the liver impaired early granuloma formation in CXCR3⫺/⫺ mice during
L. donovani infection, given that flow cytometry analysis
showed that both CXCR3+/+ and CXCR3⫺/⫺ mice recruited
comparable proportions of Gr1+ neutrophils to their livers during infection (data not shown). Nevertheless, our data show
that, although CXCR3 may be involved in the control of early
hepatic inflammation and granuloma formation after L. donovani infection, it is not essential for the recruitment of immune
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cells to the liver during the late stage of the infection. Additionally, these findings also suggest that other T cell–associated
C-C and CXC chemokine receptors may be involved in the
migration of effector CD4+ and CD8+ T cells to the liver and
spleen. One candidate is CXCR6, which interacts with CXCL16
and mediates T cell recruitment to the inflamed liver. Further
study of this chemokine–chemokine receptor interaction during VL should prove worthwhile in the exploration of liverspecific T cell migration.
There may be several reasons why a lack of CXCR3 impairs
the migration of T cells to the skin during L. major infection
but has no effect on T cell trafficking to the liver and spleen
during L. donovani infection. First, L. major and L. donovani
cause different diseases in humans and mice and trigger complex immune responses that determine the outcome of the
disease in the host. Second, it is becoming increasingly evident
that chemokine receptors play organ-specific roles in the regulation of leukocyte trafficking. For example, selective recruitment of naive lymphocytes to the secondary lymphoid organs
is mediated largely by the action of CCR7 and its ligands,
whereas CCR9 is involved in T cell development in the thymus.
Similarly, among the so-called inflammatory chemokine receptors, CCR4 and CCR10 have been implicated in the selective
recruitment of certain T cell subsets to the skin [11]. Our
findings in the present study, together with our previous findings, demonstrate clearly that, even though CXCR3 is necessary
for optimal recruitment of T cells to the skin, other chemokinedependent mechanisms efficiently recruit T cells to spleen and
liver tissue during inflammation.
IL-12–driven CD4+ Th1 lymphocytes play a critical role in
immunity to L. donovani [12]. However, the clear Th1-Th2
pattern of disease development shown for L. major is not observed in VL caused by L. donovani in mice and humans, because Th2 cytokines (such as IL-4 and IL-13) do not determine
susceptibility [13, 14]. In fact, IL-4⫺/⫺ and IL-4Ra⫺/⫺ mice are
more susceptible to L. donovani, indicating that IL-4 may play
a disease-protective role during VL [15]. Previous studies have
shown that CXCR3 plays a critical role in T cell activation.
Similarly, CXCL9 and CXCL10 also stimulate T cell proliferation and effector cytokine production [4]. We therefore analyzed
Th1 and Th2 responses in L. donovani–infected CXCR3+/+ and
CXCR3⫺/⫺ mice by determining titers of L. donovani antigen
(LdAg)–specific Th1-associated IgG2a and Th2-associated IgG1
and by measuring proliferation and cytokine production in
spleen cells after in vitro stimulation with LdAg. Additionally,
we compared levels of tumor necrosis factor (TNF)–a, IL-12,
IFN-g, IL-4, and IL-10 mRNA in infected livers by real-time
RT-PCR. Throughout the course of infection, both groups displayed similar titers of L. donovani–specific Th1-associated
IgG2a and Th2-associated IgG1 (data not shown). On days 15,
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30, and 60 after infection, LdAg-stimulated spleen cells from
L. donovani–infected CXCR3+/+ and CXCR3⫺/⫺ mice displayed
comparable proliferation responses (data not shown) and produced comparable amounts of IL-12, IFN-g, IL-4, and IL-10
(figure 2A–2D). At all time points, infected livers from both
groups contained comparable amounts of TNF-a, IL-12, IFNg, and IL-10 mRNA. Levels of IL-4 mRNA were also similar
in CXCR3+/+ and CXCR3⫺/⫺ mice on days 15 and 30; however,
CXCR3⫺/⫺mice consistently contained significantly more IL-4
mRNA in their livers on day 60 (figure 2E–2I). Additionally,
no significant difference in CXCL9 and CXCL10 mRNA levels
was found in the livers of CXCR3+/+ and CXCR3⫺/⫺ mice at
all time points assayed (data not shown). These results demonstrate that a lack of CXCR3 does not alter T cell activation,
production of Th1 and Th2 cytokines, and recruitment of IFNg–producing cells to the liver and spleen during L. donovani
infection.
In sum, although CXCR3⫺/⫺ C57BL/6 mice show delayed
formation of liver granulomas after L. donovani infection, they
eventually recruit sufficient numbers of T cells to the liver and
spleen and control L. donovani infection as efficiently as do
CXCR3+/+ C57BL/6 mice. Furthermore, resolution of L. donovani infection by CXCR3⫺/⫺ mice is associated with the development of an efficient Th1 response. These results also suggest that, although the CXCR3 ligands CXCL9 and CXCL10
are produced at high levels during L. donovani infection, they
may play a minor role in the host defense against this parasite.
Acknowledgments
We thank Sarah E. Walker and Heidi M. Snider, for critical review of
the manuscript.
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