Susceptibility to Leishmania mexicana infection is due to the inability

Immunology and Cell Biology (2001) 79, 320–322
Research Article
Susceptibility to Leishmania mexicana infection is due to the
inability to produce IL-12 rather than lack of IL-12 responsiveness
M I R I A M RO D R I G U E Z - S O S A , G I NA M M O N T E F O RT E a n d A B H AY R S ATO S K A R
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA
Summary Almost all inbred mice are highly susceptible to parasites of the Leishmania mexicana complex that
includes L. amazonensis and L. mexicana. Recent studies have reported that T cells from L. amazonensis-infected
mice fail to respond to IL-12 due to impaired IL-12R expression. Here, we demonstrate that lymph node cells from
L. mexicana-infected C57BL/6 and 129Sv/Ev mice respond efficiently to exogenous IL-12 in vitro and produce
IFN-γ. Moreover, we also show that deletion of signal transducer and activator of transcription (STAT)4 gene in
resistant STAT6–/– mice renders them susceptible to L. mexicana. These findings indicate that an inability to
produce IL-12 rather than unresponsiveness to this cytokine is responsible for susceptibility to L. mexicana. Moreover, the data also demonstrate that the STAT4-mediated pathway is critical for the development of protective
immunity against cutaneous leishmaniasis, regardless of the species of Leishmania and/or genetic background of
the mice.
Key words: Leishmania mexicana, STAT4, STAT6.
Introduction
American cutaneous leishmaniasis is caused by parasites of
the Leishmania mexicana complex that includes L. amazonensis and L. mexicana.1 It is well documented that almost all
inbred mice are susceptible to L. mexicana complex and
develop large non-healing lesions following infection with
these parasites.2,3 We have previously demonstrated that
IL-4–/– as well as signal transducer and activator of transcription (STAT)6–/– mice produce high levels of IL-12, develop a
Th1 response and control L. mexicana infection.4,5 These
findings indicate that IL-4 and the STAT6-mediated signalling pathway mediate susceptibility to L. mexicana by
suppressing IL-12 production and Th1 development. In contrast, other studies indicate that the inability to generate a Th1
response rather than the presence of IL-4 or a Th2 response
may be responsible for susceptibility to L. amazonensis.6,7
Moreover, lack of Th1 in L. amazonensis-infected mice has
been attributed to the inability of CD4+ T cells to respond to
IL-12 due to the suppression of IL-12Rβ expression.7
Therefore, in this study, we determined whether lack of
IL-12 production or inability to respond to this cytokine was
responsible for impaired Th1 development and susceptibility
to L. mexicana infection.
Materials and Methods
Animals
Six to eight-week-old female C57BL/6 and 129Sv/Ev mice were
purchased from Taconic Laboratories (Germantown, NY, USA).
Correspondence: Dr AR Satoskar, Department of Microbiology,
The Ohio State University, 484 W 12th Avenue, Columbus, OH
43210, USA. Email: [email protected]
Received 1 December 2000; accepted 26 February 2001.
STAT4/STAT6 gene-deficient C57BL/6 × 129Sv/Ev mice were generated by intercrossing STAT4–/– and STAT6–/– C57BL/6 × 129Sv/Ev
mice that were kindly provided by Dr James Ihle (St. Jude’s Hospital, Memphis, TN, USA). These mice were bred and maintained in a
facility at the Harvard School of Public Health according to the
guidelines for animal research.
Parasite and infection protocols
For all experiments, mice were infected by injecting 5 × 106 L. mexicana (M379) amastigotes into shaven back rump. The disease
progression was monitored by measuring lesion diameters weekly
until week 10 postinfection.
Antibody ELISA
Mice were bled at different time points from tail snips following
L. mexicana infection. Blood was centrifuged at 200 × g, and serum
was collected and tested for L. mexicana antigen (LmAg)-specific
Th1-associated IgG2a and Th2-associated IgG1 antibodies by ELISA
as described previously.5
T-cell proliferation and cytokine assays
At week 10 postinfection the mice were killed, their lymph nodes
excised and T-cell proliferation assays were performed as described
previously.5 Briefly, 3 × 105 lymph node cells were added to the wells
of 96-well flat-bottomed tissue culture plates and stimulated with
20 µg/mL of LmAg prepared by repeated freezing and thawing of
stationary phase promastigotes. Supernatants from these cultures
were analysed for IL-4, IL-12 and IFN-γ production by ELISA.5
Results and Discussion
It is widely accepted that IL-12 plays a critical in the development of Th1 response and protective immunity during
Susceptibility to L. mexicana infection
Figure 1 In vitro production of IFN-γ by L. mexicana antigen
(LmAg)-stimulated lymph node cells from (a) C57BL/6 and
(b) 129Sv/Ev mice following addition of recombinant murine
IL-12. Five animals were used in each group. Data are expressed
as means ± SEM and are representative of three experiments.
Figure 2 Course of cutaneous L. mexicana infection in
STAT4/STAT6+/+ (), STAT4/STAT6–/– () and STAT6–/– mice
(). (a) Lesion growth was monitored by measuring lesion diameters following L. mexicana infection. Data are expressed as
mean lesion diameter ± SE. At week 10 postinfection in vitro
L. mexicana antigen (LmAg)-induced (b) IFN-γ and (c) IL-4 by
the lymph node cells from STAT4/STAT6+/+ (wild type) and
STAT4/STAT6–/– mice were measured by ELISA. Four to five
animals were used in each group.
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cutaneous as well as visceral leishmaniasis.8,9 As demonstrated previously, C57BL/6 and 129Sv/Ev mice were highly
susceptible to L. mexicana and developed large non-healing
lesions following L. mexicana infection (data not shown). At
week 10 postinfection, LmAg-stimulated lymph node cells
from these mice failed to produce significant levels of
IFN-γ, which was associated with basal levels of IL-12
(Fig. 1). However, addition of exogenous recombinant
murine IL-12 to these cultures induced significant IFN-γ
production by LmAg-stimulated lymph node cells from
these inbred strains (Fig. 1). These results demonstrate that
T cells, regardless of the genetic background of the mouse,
can respond efficiently to IL-12 and produce IFN-γ during
L. mexicana infection. Moreover, these results also suggest
that lack of IL-12 production rather than responsiveness to
this cytokine may be responsible for impaired IFN-γ production following L. mexicana infection. It is possible that
IL-12-induced IFN-γ may be derived partly from other nonCD4+ T cells, such as NK cells. Nevertheless, these findings
differ from those observed in previous studies demonstrating
that the lack of IL-12 responsiveness resulting from impaired
IL-12Rβ expression inhibits and mediates susceptibility to
L. amazonensis.7
We previously found that genetically susceptible C57BL/
6 × 129Sv/Ev mice lacking STAT6 gene produce significantly higher levels of IL-12, develop a Th1 response and
control L. mexicana infection, suggesting that L. mexicana
infection does not alter in vivo responsiveness of T cells to
IL-12.5 If susceptibility to L. mexicana was due to the lack of
IL-12 responsiveness, STAT6–/– mice would have failed to
mount a Th1 response and remained susceptible to L. mexicana despite production of high levels of IL-12. Recent
studies have demonstrated that CD4+ and CD8+ T cells differ
in their regulation of IFN-γ.10 While STAT4-mediated IL-12R
signalling is critical for IFN-γ production in CD4+ T cells via
the TCR pathway and Th1 development, CD8+ T cells can
produce IFN-γ independently of IL-12.10 Therefore, we
hypothesized that deletion of STAT4 gene in STAT6–/– mice
will impair Th1 development and render them susceptible to
L. mexicana. As anticipated, in the present study we found
that STAT4/STAT6–/– C57BL/6 × 129Sv/Ev mice developed
rapidly progressive large non-healing lesions similar to
STAT4/STAT6+/+ mice (Fig. 2a). In contrast, concomitantly
infected STAT6–/– mice failed to develop lesions (Fig. 2a). At
week 10 postinfection, LmAg-stimulated lymph node cells
from STAT4/STAT6 mice produced significantly lower
levels of IFN-γ and IL-4 than wild-type mice did (Fig. 2b,c).
These observations demonstrate that in the absence of
STAT4-mediated IL-12 signalling pathway STAT6–/– mice
become susceptible to L. mexicana. These findings demonstrate that the IL-12R/STAT4-mediated pathway is functional
and indispensable for Th1 development in STAT6–/– mice
during L. mexicana infection. These findings support our
previous observations in a Leishmania major model,11 and
indicate that the STAT4-mediated pathway is critical for the
development of protective immunity against cutaneous leishmaniasis regardless of the species of Leishmania and/or
genetic background of the mice.
In conclusion, our data indicate that unlike L. amazonensis,
failure to develop an efficient Th1 response during L. mexicana infection is due to the lack of IL-12 production rather
than the inability of T cells to respond to this cytokine.
Moreover, the data also demonstrate that in the absence of
STAT6, the STAT4-mediated IL-12 signalling pathway is
indispensable for the development of protective immunity
against L. mexicana.
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