Article

Redundant Notch1 and Notch2 Signaling Is Necessary for IFNγ Secretion by T Helper 1 Cells During Infection with Leishmania major

Department of Biochemistry, WHO Immunology Research and Training Center, University of Lausanne, Epalinges, Switzerland.
PLoS Pathogens (Impact Factor: 7.56). 03/2012; 8(3):e1002560. DOI: 10.1371/journal.ppat.1002560
Source: PubMed
ABSTRACT
The protective immune response to intracellular parasites involves in most cases the differentiation of IFNγ-secreting CD4(+) T helper (Th) 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization of peripheral CD4(+) T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1) and Notch2 (N2) are expressed on activated CD4(+) T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection, mice with T cell-specific gene ablation of N1, N2 or both (N1N2(ΔCD4Cre)) were infected with the protozoan parasite Leishmania major. N1N2(ΔCD4Cre) mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNγ by draining lymph node CD4(+) T cells and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were resistant to infection and developed a protective Th1 immune response, showing that CD4(+) T cell expression of N1 or N2 is redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNγ by Th1 cells. This effect is independent of CSL/RBP-Jκ, the major effector of Notch receptors, since L. major-infected mice with a RBP-Jκ deletion in their T cells were able to develop IFNγ-secreting Th1 cells, kill parasites and heal their lesions. Collectively, we demonstrate here a crucial role for RBP-Jκ-independent Notch signaling in the differentiation of a functional Th1 immune response following L. major infection.

Full-text

Available from: Fabienne Tacchini-Cottier
Redundant Notch1 and Notch2 Signaling Is Necessary for
IFNc Secretion by T Helper 1 Cells During Infection with
Leishmania major
Floriane Auderset
1
, Steffen Schuster
1
, Manuel Coutaz
1
, Ute Koch
2
, Florian Desgranges
1
, Estelle Merck
3
,
H. Robson MacDonald
3
, Freddy Radtke
2
, Fabienne Tacchini-Cottier
1
*
1 Department of Biochemistry, WHO Immunology Research and Training Center, University of Lausanne, Epalinges, Switzerland, 2 Ecole Polytechnique Fe
´
de
´
rale de
Lausanne, Swiss Experimental Cancer Research, Lausanne, Switzerland, 3 Ludwig Institute for Cancer Research Ltd, Lausanne Branch, University of Lausanne, Epalinges,
Switzerland
Abstract
The protective immune response to intracellular parasites involves in most cases the differentiation of IFNc-secreting CD4
+
T
helper (Th) 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization
of peripheral CD4
+
T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1) and Notch2 (N2) are
expressed on activated CD4
+
T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection,
mice with T cell-specific gene ablation of N1, N2 or both (N1N2
DCD4Cre
) were infected with the protozoan parasite
Leishmania major. N1N2
DCD4Cre
mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions
and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNc by draining lymph node CD4
+
T cells
and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were
resistant to infection and developed a protective Th1 immune response, showing that CD4
+
T cell expression of N1 or N2 is
redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNc by
Th1 cells. This effect is independent of CSL/RBP-Jk, the major effector of Notch receptors, since L. major-infected mice with a
RBP-Jk deletion in their T cells were able to develop IFNc-secreting Th1 cells, kill parasites and heal their lesions. Collectively,
we demonstrate here a crucial role for RBP-Jk-independent Notch signaling in the differentiation of a functional Th1
immune response following L. major infection.
Citation: Auderset F, Schuster S, Coutaz M, Koch U, Desgranges F, et al. (2012) Redundant Notch1 and Notch2 Signaling Is Necessary for IFNc Secretion by T
Helper 1 Cells During Infection with Leishmania m ajor . PLoS Pathog 8(3): e1002560. doi:10.1371/journal.ppat.1002560
Editor: David L. Sacks, National Institute of Health, United States of America
Received October 3, 2011; Accepted January 17, 2012; Published March 1, 2012
Copyright: ß 2012 Auderset et al. This is an open-access article distri buted under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants to FTC from the Swiss National Science Foundation 3100030_129852, and 129700/1. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Fabienne.Tacchini-Cottier@unil.ch
Introduction
Following activation by pathogens, naı
¨
ve CD4
+
T cells can
differentiate into several functionally distinct T helper (Th) subsets,
defined by the cytokines they secrete. CD4
+
Th1 cells secrete IFNc
as a signature cytokine and the transcription factor T-bet is
essential for their differentiation. Although cytokines such as IL-12
contribute to Th1 cell differentiation, Th1 cells can be generated
in the absence of cytokine signaling, demonstrating a role for other
molecules in this process. Among these are Notch receptors and
their ligands (Reviewed in [1,2]). Notch signaling plays crucial
roles in binary cell fate decisions in many developmental systems
including the development and differentiation of immune cells. In
mammals, there are four Notch receptors (Notch1-4) that are
activated by five ligands (Jagged (Jag) 1, and 2, and Delta-like (Dll)
1, 3, and 4). Upon interaction with its ligand, the active
intracellular domain of Notch (NICD) is released from the
membrane by proteolytic cleavages and translocates into the
nucleus. Once there, NICD can form a complex with recombi-
nation signal-binding protein-J (RBP-Jk), converting it to an
activator of transcription (canonical Notch signaling). Alternative-
ly, NICD could interact with members of the NF-kB pathway
(non-canonical Notch signaling) [3]. In the T cell lineage, the
Notch1 receptor is essential for the development of ab T cells [4],
and Notch plays a poorly understood role in the differentiation of
peripheral Th cell subsets (reviewed in [1,5]).
The importance of Notch signaling during CD4
+
Th1
differentiation and its correlated resolution of pathogen infection
is currently unclear. Inhibitors of c-secretase impairing Notch
signaling prevented Th1 differentiation in vitro and in vivo,
potentially through the blocking of T-bet expression [6]. Blocking
of the Notch3 receptor using antisense N3 DNA also blocked Th1
differentiation in vitro [7]. In contrast, T cell-specific expression of
dominant negative mastermind-like protein (MAML1), which is
needed for RBP-Jk-dependent Notch signaling, or T cell specific
ablation of Notch1 or RBP-Jk did not have an impact on Th1
differentiation in vitro [8] nor in vivo [9,10].
The role of Notch ligands on dendritic cells instructing Th1
differentiation is also debated. Dll1 and/or Dll4 expression is
upregulated in vitro on APCs encountering pathogens driving a
CD4
+
Th1 response [8,11,12]. Interaction of Notch with Dll1
promoted Th1 differentiation during Leishmania major infection [7].
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Furthermore, Dll4 expression on DC was shown to induce Th1
cell differentiation in an IL-12-independent way [11]. On the
contrary, Dll1, Jag1 and Jag2 were shown to be insufficient to
instruct the differentiation of Th1 or Th2 CD4
+
cells in absence of
polarizing cytokines in vitro, suggesting that the induction of
selective ligands by pathogens may not exert a direct influence on
T helper differentiation [13,14].
Altogether, these studies indicate a role of Notch in CD4
+
Th1
differentiation, but it is not clear yet which member and how each
member of this family contributes to this process during infection
with pathogens. Most of the above studies investigated the role of
Notch using total inhibition of Notch signaling, but the individual
contribution and potential crosstalk of individual Notch receptors
during infections with pathogens inducing CD4
+
Th1 cells has not
been investigated.
Here, mice carrying a T cell specific deletion of Notch1
(N1
DCD4Cre
), Notch2 (N2
DCD4Cre
) or both Notch1 and Notch2
(N1N2
DCD4Cre
) on a resistant C57BL/6 genetic background were
infected with L. major to study the importance of Notch receptors
in Th1 differentiation and resolution of the infection. We show
that Notch signaling through either N1 and/or N2 induces the
secretion of IFNc by CD4
+
Th1 cells. Moreover, using mice with
T cell-specific ablation of RBP-Jk (RBP-J
DCD4Cre
), we show that
Th1 differentiation is induced mainly by non-canonical (RBP-Jk-
independent) Notch signaling. Collectively, our data indicate that
Notch signaling drives the differentiation of L. major-specific IFNc-
secreting Th1 cells required to mount an efficient immune
response against this parasite.
Results
Notch affects the development of a protective L. major-
specific Th1 cell response
To investigate Notch function in Th1 cell development we
infected mice with L. major, a parasite promoting a predominant
Th1 immune response in most strains of mice including C57BL/6
[15]. Of the four Notch receptors, only N1 and N2 are expressed
in activated T cells [8,16]. Thus, to investigate the effect of T cell
ablation of these two receptors (N1N2
DCD4Cre
) on CD4
+
Th1
differentiation and the consequent resolution of L. major infection,
N1N2
DCD4Cre
and control N1N2
lox/lox
mice on the L. major
resistant C57BL/6 genetic background were inoculated with the
parasite. In contrast to N1N2
lox/lox
control mice that developed a
small self-healing lesion, N1N2
DCD4Cre
were unable to heal their
lesions (Figure 1A). In addition, L. major-infected N1N2
DCD4Cre
mice failed to control parasite load at the site of parasite
inoculation (Figure 1B) and L. major disseminated to the lymph
nodes and spleen (Figure 1C, D).
The impact of the absence of Notch on T cells in the
differentiation of CD4
+
IFNc-secreting Th1 cells was assessed six
weeks after infection. N1N2
DCD4Cre
and control draining lymph
node (dLN) cells were restimulated in vitro with UV-irradiated L.
major and cytokine levels measured. Strikingly, secretion of IFNc
was abrogated in supernatants of N1N2
DCD4Cre
dLN cells, while
high levels of this cytokine were measured in dLN from infected
control mice (Figure 1E). Similarly low IL-4 levels were measured
in each group (Figure 1F). IL-13 and IL-5 were found
predominantly in N1N2
DCD4Cre
dLN cells (Figure 1G). The
persistence of parasites in the dLN of L. major infected
N1N2
DCD4Cre
but not N1N2
lox/lox
mice was sufficient to induce
IL-13 and IL-5 secretion by T cells (Figure 1G), albeit at a lower
level than that reached following stimulation with exogenous
addition of L. major. These results show that Notch signaling
contributes to the generation of IFNc-secreting CD4
+
T cells,
which are essential in the control of parasite load and lesion size.
The absence of Notch expression on T cells, while preventing
IFNc secretion, favored the development of IL-13- and IL-5-
secreting cells.
N1 or N2 expression on T cells is sufficient to drive Th1
cell differentiation
N1
DCD4Cre
mice are able to develop a protective Th1 response
in response to L. major inoculation [9]. The inability of L. major -
infected N1N2
DCD4Cre
mice to develop a protective Th1 immune
response suggested that N2 could be the receptor involved in Th1
differentiation. To investigate this, N2
DCD4Cre
and N2
lox/lox
control
mice were infected with L. major and evolution of lesion size and
development of immune response were compared to that
developing in N1
DCD4Cre
, and N1N2
DCD4Cre
infected mice.
N2
DCD4Cre
mice were able to control their lesion size (Figure 2A)
and parasitemia (Figure 2B) as well as N1
DCD4Cre
and control
mice, unlike N1N2
DCD4Cre
mice. To analyze their immune
response, cytokine secretion by dLN cells was analyzed. L. major-
infected N2
DCD4Cre
dLN cells secreted similar levels of IFNc than
L. major-infected N1
DCD4Cre
and control mice. Low levels of IL-4,
IL-5 and IL-13 were similarly measured in their dLN cells
(Figure 2C).
N1N2
DCD4Cre
mice are susceptible to L. major infection and fail
to induce IFNc secretion by CD4
+
T cells, indicating that
expression of either N1 or N2 is sufficient to induce CD4
+
Th1
differentiation in N2
DCD4Cre
or N1
DCD4cre
mice, respectively. To
investigate if following parasite inoculation, compensatory expres-
sion of one or all of the Notch receptors on T cells could occur,
dLN cells of L. major infected N1
DCD4Cre
or N2
DCD4Cre
mice were
stimulated in vitro with L. major, and Notch expression on their
CD4
+
T cells measured by FACS. N1 expression was significantly
and similarly expressed in both N2
DCD4Cre
and N2
lox/lox
CD4
+
T
cells (Figure 3A). Low levels of N2 surface expression were induced
following restimulation of control dLN T cells with L. major,
however, a significantly higher induction of N2 was measured in L.
major-activated N1
DCD4cre
CD4
+
T cells (Figure 3B), suggesting
that compensatory mechanisms allow increased N2 expression in
Author Summary
Infection with protozoan parasites of Leishmania species
results in a spectrum of local or systemic diseases in
humans and mammals. Overall, leishmaniasis afflicts
around 12 million individuals in 88 countries worldwide.
Cutaneous leishmaniasis is the most prevalent form of the
disease. In order to better understand the complex
molecular pathways leading to protection against the
cutaneous form of the disease, we used the Leishmania
major mouse model. Most mouse strains control L. major
infection due to the development of a Th1 response,
leading to secretion of IFNc by T cells which promotes
healing and resistance to reinfection. Notch signaling is a
very conserved pathway in the regulation of cell differen-
tiation and cell fate decision. However the contribution of
Notch receptors in the response to parasite infection is not
clear. In this study, we infected mice that do not express
Notch1 and Notch2 receptors on the surface of their T
cells. We show that these Notch receptors are key players
in the development of a protective Th1 immune response
against L. major. These results contribute to the under-
standing of the mechanisms involved in the development
of a protective response against pathogens.
Notch Receptors Regulate T Cell IFNc Secretion
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Page 2
absence of N1. No expression of N3 and N4 mRNA or proteins
was detectable on T cells of all genotypes, in contrast to positive
control cells (Figure 3C and data not shown). Altogether, these
data reveal that signaling through either N1 or N2 is sufficient for
the generation of functional Th1 cells following infection with L.
major, and that in absence of N1 compensatory higher expression
of N2 is measured on T cells.
Susceptibility to L. major infection in absence of Notch
on T cells results mainly from lack of IFNc secretion
Draining LN CD4
+
T cells of L. major-infected N1N2
DCD4Cre
mice fail to secrete IFNc but release high levels of IL-13. This
cytokine has been associated with susceptibility to L. major
infection, mostly by preventing the classical activation of
macrophages by IFN c [17]. To investigate if susceptibility of
N1N2
DCD4Cre
mice resulted from a lack of IFNc secretion and/or
from the presence of high levels of IL-13, IL-13 was neutralized
with an anti-IL-13 mAb after inoculation of L. major in
N1N2
DCD4Cre
and N1N2
lox/lox
control mice. No effect was
observed in lesion development and parasite control in IL-13-
depleted N1N2
DCD4Cre
mice, that developed unhealing lesions
similar to mice treated with PBS (Figure 4A). Similar low levels of
IFNc were measured in isolated CD4
+
T cells of mice depleted or
not of IL-13 (Figure 4B). The efficacy of the anti-IL-13 treatment
was confirmed by measuring dLN levels of Fizz1 and Ym1
expression, two markers of alternative macrophage activation. The
mRNA levels of both markers were decreased in dLN of anti-IL-
13-treated N1N2
DCD4Cre
mice (Figure 4C). Collectively, these data
demonstrate that the non-healing phenotype measured in
N1N2
DCD4Cre
mice results primarily from the decreased IFNc
Figure 1. N1N2
DCD4Cre
mice on the C57BL/6
L. major
resistant background are susceptible to infection. (A) N1N2
DCD4Cre
and control
N1N2
lox/lox
mice were infected with 3610
6
L. major promastigotes and lesion size measured weekly. Dots represent group mean of lesion size 6 SEM.
(B, C) Six weeks after infection, parasite load was assessed by LDA in footpads (B), dLN (C) and spleen (D). Histograms represent the mean number of
parasite 6 SEM (n$3 mice per group). (E–G) IFNc (E), IL-4 (F), IL-13 and IL-5 (G) secretion was quantified in supernatants of draining lymph node cells
restimulated or not with UV-irradiated L. major 6 weeks after infection. Mean cytokine secretion 6 SEM are given (n$3 mice per group). Data are
representative of at least 3 individual experiments. n.d. not-detectable. * p-value,0.05 versus control mice.
doi:10.1371/journal.ppat.1002560.g001
Notch Receptors Regulate T Cell IFNc Secretion
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Page 3
secretion by CD4
+
T cells which does not allow activation of
macrophage to kill the intracellular parasites. The high levels of
IL-13 which induce alternative macrophage activation do not play
a critical role in the failure of macrophages to kill the parasites, as
in absence of IFNc, macrophages are already not classically
activated.
Notch signaling prevents the release but not the
transcription of IFNc by CD4
+
dLN cells
We then investigated if the impaired IFNc secretion measured
in N1N2
DCD4Cre
CD4
+
T cells could result from defective in vitro
proliferation. To this end, dLN cells of L. major-infected
N1N2
DCD4Cre
and control mice were stained with CFSE and
restimulated for 72 h with L. major. CD4
+
T cells of both
N1N2
DCD4Cre
and control proliferated in response to the parasite.
N1N2
DCD4Cre
showed a slightly lower CD4
+
T cell proliferation
compared to that of N1N2
lox/lox
CD4
+
T cells but the difference
was not statistically significant (Figure 5A). Despite comparable
proliferation, IFNc was not secreted in response to L. major
stimulation. However, high levels of intracellular IFNc were
measured by FACS in N1N2
DCD4Cre
CD4
+
T cells restimulated
with L. major for 72 h, in absence of PMA-ionomycin stimulation
(Figure 5B). To further determine at which level the absence of N1
and N2 on T cells affects secretion of IFNc in the dLN of L. major-
infected mice, mRNA levels of IFNc and T-bet, the major
transcription factor of Th1 cells, were measured ex vivo on FACS
sorted CD4
+
T cells 3 weeks after infection. Reduced levels of
secreted IFNc did not result from impaired transcription of T-bet
or IFNc mRNA as demonstrated by higher levels of both T-bet
and IFNc mRNA measured in N1N2
DCD4Cre
CD4
+
T cells
compared to those measured in CD4
+
T cells of control mice
(Figure 5C). No defect in activation status or in CD4
+
T cell
number was measured in dLN cell N1N2
DCD4Cre
mice (Figure S1).
IFNc signaling is mediated by STAT1 phosphorylation and IFNc
was reported to signal to the majority of cells throughout the dLN
during a Th1 response after T. gondii infection [18]. To further
show that secretion of IFNc is impaired in CD4
+
T cells during
infection, we measured STAT1 phosphorylation in dLN CD4
+
T
cells of L. major-infected N1N2
DCD4Cre
mice. STAT1 phosphor-
ylation was detected in CD4
+
T cells of control mice while only
Figure 2. N1 and N2 alone can drive CD4
+
Th1 differentiation. (A) N1
DCD4Cre
,N2
DCD4Cre
, N1N2
DCD4Cre
, and control mice were infected with
3610
6
L. major promastigotes and lesion size measured weekly. Data are represented as the mean of lesion size 6 SEM with n$3 mice per group. (B)
Parasite load in the lesion was assessed by LDA 6 weeks after infection. Mean parasite number is given 6 SEM (n$3 mice per group) (C, D) Six weeks
after infection, IFNc (C), IL-4 and IL-13 (D) secretion was assessed in supernatant of dLN cells restimulated or not with UV-irradiated L. major for 72 h.
Histograms show the mean cytokine secretion 6 SEM (n$3 mice per group). n.d. not-detectable, n.s. not significant. * p-value,0.05 versus control
mice.
doi:10.1371/journal.ppat.1002560.g002
Notch Receptors Regulate T Cell IFNc Secretion
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minimal STAT1 phosphorylation was measured in CD4
+
T cells
of N1N2
DCD4Cre
dLN cells (Figure 5D). These data confirm that in
absence of N1 and N2 on T cells, IFNc secretion by CD4
+
T cells
is impaired, thereby preventing IFNc-induced STAT1 phosphor-
ylation occurring in vivo. The impairment of IFNc secretion is
antigen-specific and not due to an intrinsic secretion default in
N1N2
DCD4Cre
mice as revealed by the high levels of IFNc detected
by intracellular staining following TCR-independent T cell
stimulation (PMA-ionomycin) ex vivo (Figure 5E). In addition,
defective secretion could be overcome in vitro by antibody-
mediated CD3 crosslinking stimulation (Figure S2A).
In the same line, naı
¨
ve N1N2
DCD4Cre
CD4
+
T cells stimulated in
vitro in the presence of standard Th1 polarizing conditions followed
by stimulation with anti-CD3/CD28 for 48 hours were able to
develop into IFNc-secreting cells (Figure S2B). In contrast, the
strong increase in IL-13 and IL-5 mRNA levels measured in dLN
of L. major -infected mice (Figure 5F) correlated with the high
secretion levels of these cytokines in L. major-stimulated CD4
+
T
cells. Altogether, these data reveal that following inoculation of L.
major, absence of N1 and N2 on T cells prevents the release of
IFNc by CD4
+
T cells, favoring the differentiation of IL-13- and
IL-5-secreting cells. This effect is obscured in vitro by exogenous
addition of high amounts of cytokines and/or antigen-non specific
activation of T cells.
Notch signaling driving CD4
+
Th1 differentiation occurs
in absence of the RBP-Jk transcription factor
Mice with dominant negative MAML (DNMAML) protein
preventing the canonical transcriptional activation by all four
Notch receptors were previously reported to be able to control
infection with L. major and to have normal levels of IFNc in their
dLN CD4
+
T cells [10]. Different strains of L. major may induce
distinct type of T helper immune response [19]. To further insure
that the different outcomes on the differentiation of Th1 cells
measured in theirs (L. major Friedlin) and the present studies (L.
major LV39) were not due to differences in the L. major strains used,
N1N2
DCD4Cre
mice were infected with two other L. major strains
(Friedlin or IR175). N1N2
DCD4Cre
mice infected with these two L.
major strains failed to develop an efficient Th1 response with
decreased secretion of IFNc and increased secretion of IL-13 and
IL-5 by their dLN T cells and high intralesional parasite load
(Figure S3). These data show that N1 and N2 are required for Th1
differentiation following infection with different strains of L. major.
Having ruled out a potential effect due to the strain of Leishmania
used, the lack of effect of DNMAML on Th1 differentiation [10]
suggested that Notch signaling may not drive Th1 cell differen-
tiation through the NICD–MAML–RBP-Jk transcriptional acti-
vation complex. To investigate if the requirement of Notch
signaling for CD4
+
Th1 differentiation and the associated
resolution of the lesion could be RBP-Jk-independent, we infected
RBP-Jk
DCD4Cre
and RBP-Jk
lox/lox
control mice with L. major.No
difference in lesion development (Figure 6A) nor parasite control
was measured between RBP-Jk
DCD4Cre
and control mice
(Figure 6B). Furthermore, the development of CD4
+
IFNc-
secreting Th1 cells was normal, as revealed by high levels of
IFNc secretion by dLN T cells, and low levels of IL-13 and IL-5
(Figure 6C). These results demonstrate that RBP-Jk-independent
Notch signaling is required for CD4
+
Th1 differentiation following
L. major infection.
Discussion
The development of Th1 cells, through their secretion of IFNc,
contributes to a number of protective effects against many
pathogens. Despite a growing understanding on the mechanisms
leading to T helper differentiation these last years (reviewed in
[20]), there are still unresolved issues including the identification of
which receptor triggering leads to Th1 differentiation. The role of
Notch in Th1 differentiation has been controversial, depending on
the mode of activation/deactivation of Notch [1,5]. Our data
showing the crucial role of RBP-jk-independent Notch signaling in
Figure 3. Increased N2 expression can compensate the absence
of N1 on
L. major
stimulated CD4
+
T cells. (A–C) Three weeks after
L. major infection, dLN cells from the indicated mouse strains were
isolated and restimulated for 16 h with UV-treated L. major. Notch1 (A),
Notch2 (B), Notch3 and Notch4 (C) expression by CD4
+
T cells was
assessed by FACS. CD11c
+
CD8a
+
splenic dendritic cells and
CD4
2
CD8
2
CD25
+
thymocytes were stained as positive controls for
Notch3 and Notch4 respectively. Representative flow cytometry plots
are shown. Numbers in plots represent mean fluorescence intensity MFI
6 SEM of $3 mice per group. Data are representative of 3 independent
experiments.
doi:10.1371/journal.ppat.1002560.g003
Notch Receptors Regulate T Cell IFNc Secretion
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Page 5
the differentiation of IFNc-secreting T cells help to reconcile
discrepant results obtained using different loss or gain of function
approaches that attributed or not a role for Notch signaling in Th1
differentiation.
Here, using mice with ablation of Notch in their T cells, we
demonstrate that expression of either N1 or N2 on T cells is
necessary and sufficient for the differentiation of IFNc-secreting
Th1 cells and the consequent control of infection. N1N2
DCD4Cre
mice infected with L. major failed to mount a protective Th1
immune response while mice with single deletion of N1 (this study
and [9]) or N2 in their T cells developed a protective Th1 immune
response. In control mice, N1 is the only receptor expressed at
significant levels at the surface of anti-CD3 and L. major-activated
CD4
+
T cells. There appears to be functional redundancy of N1
and N2 in driving CD4
+
T helper 1 differentiation. Expression of
N2 is low in activated T cells of L. major-infected control mice, but
compensatory increased N2 expression was measured in absence
of N1 expression. This suggests that N1 is the main receptor
involved in signaling leading to the induction of IFNc secretion by
T cells following infection with L. major, but that in absence of N1,
higher levels of N2 can compensate its absence. Functional
redundancy of N1 and N2 was previously suggested in N1IAS
mice that had decreased but not abrogated IFNc secretion [6],
however the expression of Notch receptors was not assessed in that
study. Of note, we show here that N2 is the only receptor that
could functionally substitute for N1 in driving Th1 differentiation
in vivo, and T cell expression of N3 or N4 were not detected in
presence or absence of N1 and/or N2 in CD4
+
T cells of L. major-
infected mice. In addition, N1N2
DCD4Cre
mice do not control
infection revealing that N3 and N4 are not functionally redundant
in driving IFNc secretion by CD4
+
T cells. Overexpression of N3
intracellular domain (N3IC) in T cells was previously reported to
induce IFNc secretion in vitro following anti-CD3 activation, while
overexpression of N1IC did not, suggesting that N3 could be
involved in Th1 differentiation [7]. Together with our reported
increased expression of N2 in absence of N1, these results show
that individual Notch receptors may potentially drive IFNc
secretion by CD4
+
T cells, but during L. major infection N1, and
to a lesser extent N2 appear to be the only receptors involved in
driving Th1 differentiation.
It was reported that N1 could regulate Th1 cell differentiation
by interacting with CSL sequences present in the promoter of the
Figure 4. Treatment with anti-IL-13 does not restore resistance of N1N2
DCD4Cre
mice to
L. major
. (A) N1N2
DCD4Cre
and control N1N2
lox/lox
mice were infected s.c. with 3610
6
L. major promastigotes. At day 21 of infection, N1N2
DCD4Cre
mice were treated i.p. with either anti-IL-13 mAb or
PBS as control. Treatment was repeated once a week until the end of the experiment, and lesion development was monitored. Group means of lesion
size 6 SEM (n$3 mice per group) are represented. The parasite load at the site of infection was assessed by LDA 47 days post infection. Group means
of parasite number are given 6 SEM (n$3 mice per group). (B) CD4
+
T cells were isolated by MACS from dLN of L. major-infected mice 47 days post
infection and restimulated with or without UV-treated L. major in presence of irradiated syngenic splenocytes. IFNc level was measured in
supernatant after 72 h of stimulation. Data are expressed as the group mean 6 SEM of cytokine measurement of n$3 draining lymph nodes (C) Ym1
and Fizz1 mRNA expression was analyzed in dLN cells by quantitative real-time PCR and normalized to HPRT mRNA expression. Results are
represented as fold-increase in mRNA levels relative to levels measured in control mice 6 SEM (n$3 mice per group). Data are representative of three
independent experiments. Similar results were obtained when anti-IL-13 was administrated 7 days post infection (data not shown). * p-value,0.05
versus control mice.
doi:10.1371/journal.ppat.1002560.g004
Notch Receptors Regulate T Cell IFNc Secretion
PLoS Pathogens | www.plospathogens.org 6 March 2012 | Volume 8 | Issue 3 | e1002560
Page 6
Tbx21 gene which codes for T-bet, the master regulator of Th1 cell
differentiation [6]. However, in another study, Notch was not
found to reside at the Tbx21 promoter [21]. In addition, we show
here that mice with specific ablation of RBP-Jk in their T cells,
unlike N1N2
DCD4Cre
mice, are able to mount a Th1 response and
heal their lesion following infection with L. major. These results
show that the Notch signaling playing a major role in the
differentiation IFNc-secreting cells following infection with L. major
occurs in absence of a CSL/RBP-Jk-transcription complex. In line
with these data, it was previously reported that mice that
conditionally expressed a dominant negative MAML protein
(DNMAML) and thereby are deprived of RBP-Jk-mediated
transcriptional activation of all Notch receptors, were able to
develop a protective Th1 immune response following L. major
infection [10]. The present results show that Notch receptors are
crucial to trigger secretion of IFNc by CD4
+
T cells in a CSL/
RBP-Jk-independent manner.
The nature of a CSL/RBP-Jk-independent Notch pathway is
complex and not yet defined [22]. It has been reported that Notch
can associate with the nuclear factor kB (NF-kB) proteins p50 and
Figure 5. N1N2
DCD4Cre
mice transcribe T-bet and IFNc in dLN CD4
+
T cells but do not secrete it. (A) Proliferation of CD4
+
T cells was
assessed by FACS. Draining LN cells of L. major-infected mice were isolated 6 weeks after infection, stained with CFSE and restimulated with UV-
treated L. major for 72 h. Representative flow cytometry plots gated on CD4
+
T cells are shown. Numbers in plots represent the mean percentage of
proliferating cells 6 SEM for 5 mice. (B) Intracellular levels of IFNc were analysed by FACS in L. major-infected dLN cells restimulated for 72 h with UV-
treated L. major. Representative flow cytometry plots are given. Numbers in plots represent the mean percentage of IFNc
+
cells within CD4
+
T cells 6
SEM for 5 mice. (C) Draining LN CD4
+
T cells from N1N2
DCD4Cre
and N1N2
lox/lox
mice were sorted by FACS 21 days post L. major infection, T-bet and
IFNc mRNA levels were analyzed by quantitative RT-PCR. Data are represented as the mean 6 SEM mRNA transcript levels normalized to HPRT mRNA
levels (n$3 mice per group). (D) Phosphorylation of STAT1 was assessed by FACS on dLN cells of N1N2
DCD4Cre
and N1N2
lox/lox
mice 3 weeks post
infection. Naive mice were used as control. Representative flow cytometry plots gated on CD4
+
T cells are shown. Numbers in quadrants indicate the
mean frequency of pSTAT1
+
CD4
+
T cells 6 SEM. pSTAT1 mean fluorescence intensity MFI 6 SEM is shown (n$3 mice per group). (E) Draining LN cells
of L. major-infected mice were restimulated ex vivo with PMA/ionomcyin for 4 h and level of intracellular IFNc was assessed by FACS. The frequency
of CD4
+
IFNc
+
T cells is given 6 SEM for n$3 mice per group. (F) mRNA expression of IL-13 and IL-5 was analyzed by quantitative real-time PCR in dLN
cells isolated from N1N2
DCD4Cre
and N1N2
lox/lox
mice 6 weeks post L. major infection. Results are given as mean mRNA expression relative to HPRT 6
SEM for n$3 mice per group. Data are representative of 2–3 individual experiments. * p-value,0.05 versus control mice.
doi:10.1371/journal.ppat.1002560.g005
Notch Receptors Regulate T Cell IFNc Secretion
PLoS Pathogens | www.plospathogens.org 7 March 2012 | Volume 8 | Issue 3 | e1002560
Page 7
p65. Furthermore, Notch1-NF-kB complexes could be immuno-
precipitated from the Ifng promoter despite the lack of consensus
binding sites for RBP-Jk in the promoter of this gene [23]. This
suggested that N1ICD could contribute to Th1 differentiation in a
RBP-Jk-independent way leading to the hypothesis of a connec-
tion between Notch, NF-kB and Th1 differentiation [1,5]. Of
note, NF-kB p50 is required for optimal Th1 development and L.
major-infected NF-kB1 knockout mice show a susceptible pheno-
type associated with defective secretion of IFN c [24]. However in
that study, failure to secrete IFNc was linked to a major defect in
CD4
+
T cell proliferation measured in vitro, while we did not detect
such impairment of CD4
+
T cell proliferation in Notch deficient
CD4
+
T cells. Thus Notch may interact with distinct transcription
factors involved in the secretion of IFNc by Th1 cells and further
molecular studies will be needed to determine the nature of these
factors as well as the molecular mechanisms involved in the RBP-
Jk-independent Notch signaling during Th1 differentiation.
Notch signaling is required for proper secretion of IFNc by
CD4
+
Th1 cells following antigen-specific stimulation. Interest-
ingly, increased expression of T-bet and IFNc mRNA was
measured in dLN CD4
+
T cells of L. major-infected N1N2
DCD4Cre
mice revealing that Notch signaling does not prevent the
differentiation of ‘‘competent’’ CD4
+
Th1 cells [25], but appears
to act downstream of it. The increase in T-bet and IFNc mRNA
measured in CD4
+
N1N2
DCD4Cre
T cells suggests that intact
Notch signaling regulates the extent transcription for these genes in
vivo. Low levels of STAT1 phosphorylation in dLN CD4
+
T cells
confirmed that only very small amounts of IFNc protein, maybe
released by NK cells, are present in the dLN of L. major-infected
N1N2
DCD4Cre
mice. In absence of IFNc, mice on the resistant
C57BL/6 genetic background develop a Th2 immune response,
with high levels of IL-4, IL-5 and IL-13 cytokines [26].
Accordingly, impaired secretion of IFNc by CD4
+
T cells of L.
major-infected N1N2
DCD4Cre
mice allowed the differentiation of IL-
5- and IL-13-secreting Th2 cells. However, no increased secretion
of IL-4 was measured in CD4
+
T cells of N1N2
DCD4Cre
L. major-
infected mice, in line with the previously reported crucial
importance of Notch in driving IL-4 secretion by CD4
+
T cells
[8,21,27]. Interestingly, absence of Notch did not impair the
differentiation of other Th2 effector T cells, suggesting that
following L. major infection, Notch is acting directly on the IL-4
promoter, as previously reported [8], and does not affect the
differentiation of IL-13- and IL-5-Th2 secreting cells.
Notch signaling is resulting from an interaction between Notch
receptors and ligands on antigen presenting cells. Several ligands
have been linked to Th1 differentiation in distinct experimental
models of disease and Delta-like ligands have been linked to Th1
differentiation or impaired Th2 differentiation [8,11,12,28,29]. Dll1
stimulation was shown to trigger Th1 development following L.
major infection, but it was not determined which Notch receptor was
interacting with this ligand [7]. The present study shows that either
N1 or N2 could be interacting with Dll1. Whether other Notch
ligands are involved in Notch signaling during Leishmania infection
remains to be investigated. Interestingly, it was reported recently
that within the 6q27 gene cluster, the Dll1 gene was linked to
susceptibility to visceral leishmaniasis, and reduced Dll1 expression
Figure 6. The impact of Notch signaling on Th1 differentiation is RBP-Jk-independent. (A) RBP-Jk
DCD4Cre
and RBP-Jk
lox/lox
mice were
infected s.c. with 3610
6
L. major and lesion size measured weekly. Group mean of lesion size 6 SEM for n$3 mice per group is shown. (B) Parasite
load in footpad was analyzed by LDA 5 weeks post infection. Data represent mean parasite number 6 SEM for n$3 mice per group. (C, D) IFNc (C), IL-
4, IL-13 and IL-5 (D) levels were measured in supernatant of dLN cells isolated from L. major-infected mice 5 weeks post infection and restimulated for
72 h with or without UV-treated L. major. Group mean of cytokine secretion 6 SEM is given (n$3 mice per group). n.s. not significant. * p-value,0.05
versus control mice.
doi:10.1371/journal.ppat.1002560.g006
Notch Receptors Regulate T Cell IFNc Secretion
PLoS Pathogens | www.plospathogens.org 8 March 2012 | Volume 8 | Issue 3 | e1002560
Page 8
was measured in VL patients in Sudan, Brazil, and Northen India
[30]. Thus genetic regulation of one of the Notch ligand, such as the
downregulation of Dll1 expression appears to have major
consequences on susceptibility to VL. Together with the present
study, it reveals that a proper regulation of the Notch signaling
pathway during infection with Leishmania parasites is essential for the
development of a protective response against these parasites.
Further understanding of the mechanisms by which Notch
receptors regulate the differentiation of IFNc-secreting Th1 cells
as well as the ligands involved in this process should contribute to
the development of new vaccines and immunotherapeutic targets
towards Leishmania pathology, as well as in other infections
requiring protective IFNc-secreting CD4
+
Th1 immune response.
Materials and Methods
Ethics statement
This study was carried out in strict accordance with the
recommendations in the Guide for the care and use of laboratory
animals from the Department of security and Environment (DSE)
from the state of Vaud, Switzerland. The protocol has been
approved by the Ethics and Veterinary office regulations of the
state of Vaud (SAV), Switzerland. Our laboratory has the
administrative authorization numbers 1266-3, -4 and -5.
Mice
The following T cell specific gene-targeted mice were generated
by crossing floxed Notch1 [31], floxed Notch2 [32], double floxed
Notch1-Notch2 or floxed RBP-Jk [33] mice, with mice carrying
the CD4Cre transgene [34]. N1
lox/lox
,N2
lox/lox
, N1N2
lox/lox
and
RBP-Jk
lox/lox
littermates were used as controls. All mice were on a
C57BL/6 genetic background. T cell-specific deletion of Notch
and RBP-Jk was verified for each strain by PCR. All mice were
bred and maintained under pathogen-free conditions in the animal
facility at the CIIL, Epalinges, Switzerland.
Parasites and infections
Leishmania major LV39 (MRHO/Sv/59/P strain) was used. Mice
were infected s.c. with 3610
6
stationary phase L. major promas-
tigotes in the footpad. Parasite load was assessed by limiting
dilution analysis (LDA). Treatment with CNTO 134, a rat anti-
mouse IL-13 mAb [35], a gift from Centocor, Inc, was initiated
either six days or 21 days after infection, with injection of 500
mg
i.p., once weekly in N1N2
DCD4Cre
mice, while a control group
similarly infected was treated with control IgG or PBS. As no
biological differences were observed between L. major-infected
mice treated with control IgG or PBS, PBS was used as control
vehicle for CNTO 134.
Lymphocyte culture and cytokine assays
Draining lymph node cells were cultured 6 UV-irradiated L.
major promastigotes or anti-CD3 (clone 145-2C11, eBioscience) for
72 h. CD4
+
T cells were isolated by MACS (Miltenyi Biotec), and
cultured in the presence of irradiated C57BL/6 splenocytes. For in
vitro experiment, naı
¨
ve CD4
+
CD62L
+
T cells were isolated by
MACS and cultured as previously described [9]. The cytokine
content of the cell supernatant was measured by ELISAs. IFNc
with a limit of detection of 10 IU/ml. IL-4, IL-5 (OptEIA from
BD Biosciences) and IL-13 (DuoSet from R&D Systems) cytokines
were analyzed with commercial kits.
mRNA extraction and Real-Time PCR
Extraction of total RNA was performed as previously described
[36]. Quantitative Real-Time PCRs were done using SYBR
Green and a LightCycler system (Roche). Each cytokine mRNA
was normalized to the relative hypoxanthine phosphoribosyltrans-
ferase (HPRT) endogenous mRNA expression, and represented as
arbitrary units as described previously [36]. Primers used were
previously described [36,37,38].
Flow cytometry
Draining lymph node cells were isolated 3 weeks after L. major
infection. Phosphorylation of STAT1 at tyrosine 701 (pY701) was
detected by intracellular staining using an Alexa Fluor 488
conjugated anti-Stat1, PhosFlow Fix Buffer I and Perm Buffer III
(BD Biosciences) according to manufacturer’s instructions. CD4-
PE-Cy5 and CD44-APC (eBiosciences) were used to stain cell
surface. To assess Notch receptor expression, dLN cells were
isolated and restimulated with UV-irradiated L. major for 16 hours.
Cells were stained with anti-N1, anti-N2 biotinylated mAbs [16],
followed by Streptavidin-PE, -APC (eBiosciences), PE-conjugated
anti-N3 and anti-N4 (Biolegends). CD4
2
CD8
2
CD25
+
thymocytes
were used as positive control for N3 staining, and splenic
CD8a
+
CD11c
+
dendritic cells were used as positive control for
N4 staining. CD4
+
T cells were gated using TCRb-APC and
CD4-FITC (eBiosciences) mAbs. Dead cells were excluded using
7AAD (BD Pharmingen). For T cell proliferation, dLN cells were
isolated 6 weeks post L. major infection and stained with CFSE
(Molecular Probes). Cells were then restimulated 6 UV-irradiated
L. major promastigotes for 72 h and analyzed by FACS. The
following monoclonal Ab conjugates were used: CD4-PE-Cy5,
CD8-APC, B220-Pe-TexasRed (eBioscience) and dead cells were
excluded with DAPI. Intracellular IFNc was analyzed in dLN cells
isolated 6 weeks post infection and restimulated with PMA (50 ng/
ml), ionomycin (500 ng/ml) and BrefelinA (1
mg/ml) for 4 h. Cells
were stained for surface marker with the following mAb
conjugates: CD4-PE-Cy5, CD8-APC (eBiosciences). Intracellular
IFNc-PE was detected with an anti-IFNc-PE (BD Pharmingen).
All analyses were performed on a FACS Calibur or a LSR II
(Becton Dickinson) flow cytometers and data processed with
FlowJo (TreeStar).
Statistical analysis
Data were analyzed using the Student’s t-test for unpaired data.
Supporting Information
Figure S1 CD4
+
T cell activation is not affected by
absence of Notch receptors. (A) Draining LN cells of
N1N2
DCD4Cre
and N1N2
lox/lox
mice were isolated 6 weeks post
L. major infection. Total number of cells, frequency and number of
CD4
+
T cells within dLNs are given 6 SEM (n$3 mice per
group). (B) CD44 and CD62L expression was assessed by FACS
on CD4
+
T cells 6 weeks post infection. Representative FACS
plots are shown. Histograms and numbers in quadrants represent
the mean frequency of respective cells within CD4
+
T cells 6
SEM. n.s. not significant. * p-value,0.05 versus control mice.
This is a representative experiment of three.
(EPS)
Figure S2 Strong antigen-independent stimulation of
CD4
+
N1N2
DCD4Cre
cells can promote their IFNc release
in vitro. (A) Draining LN cells of N1N2
DCD4Cre
and N1N2
lox/lox
mice were isolated 6 weeks post L. major infection and restimulated
with anti-CD3 for 72 h and the IFNc secretion was analyzed in
supernatants by ELISA. The mean IFNc secretion 6 SEM is
given for n$3 mice per group. (B) Naı
¨
ve CD4
+
CD62L
+
T cells
were isolated by MACS and differentiated in vitro under unbiased,
Th1 or Th2 condition. IFNc level was measured in supernatant by
Notch Receptors Regulate T Cell IFNc Secretion
PLoS Pathogens | www.plospathogens.org 9 March 2012 | Volume 8 | Issue 3 | e1002560
Page 9
ELISA. A representative experiment of five is shown. n.d. not
detectable. * p-value,0.05 versus control mice.
(EPS)
Figure S3 Impaired Th1 cell development in
N1N2
DCD4Cre
mice is not dependent on the L. major
parasite strain. N1N2
DCD4Cre
and control N1N2
lox/lox
mice
were infected s.c. with 3610
6
L. major promastigotes of L. major
Friedlin (A) or IR75 (B) and LV39 (C) strains. 6 weeks after
infection, CD4
+
T cells were isolated, restimulated or not with
UV-irradiated L. major Friedlin (A), IR75 (B) or LV39 (C) in
presence of irradiated spleen cells, and 72 hours later, IFNc and
IL-13 secretion was quantified in supernatants. Mean cytokine
secretion 6 SEM are given (n$3 mice per group). (D) The
intralesional parasite load was assessed by LDA 6 weeks post
infection with L. major Friedlin and IR75. Mean number of
parasite per lesion is represented 6 SEM for n$3 mice per group.
Data are representative of at least 3 individual experiments. * p-
value,0.05 versus control mice.
(EPS)
Acknowledgments
We thank, Dr Markus Mohrs for advice with the STAT1 assay and
discussions, Dr P. Branigan (Centocor) for the anti-IL-13 mAb and
discussions, Gre´goire Simon for analysis of alternate macrophage
activation, Dr Emma Fiorini and Anne Wilson for discussion, Dr Esther
von Stebut for the L. major (Friedlin), Yazmin Hauyon-La Torre, and
Tatiana Proust for technical expertise, Danny Labes for Cell sorting.
Author Contributions
Conceived and designed the experiments: FTC. Performed the experi-
ments: FA SS MC FD EM. Analyzed the data: FA FTC. Contributed
reagents/materials/analysis tools: UK FR HRM. Wrote the paper: FTC.
Contributed to the design of the experiments: FA. Contributed to the
editing of the manuscript: FA HRM FR.
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Notch Receptors Regulate T Cell IFNc Secretion
PLoS Pathogens | www.plospathogens.org 10 March 2012 | Volume 8 | Issue 3 | e1002560
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    • "The results of our allogeneic T cell stimulation assays demonstrate that notch signaling in DCs and T cells contributes to shaping the outcome of T helper cell response to pollen. Blocking of notch signaling was previously shown to inhibit Th1 differentiation in vitro and in animal models [16,39,40]. DAPT works by inhibiting gamma secretase and therefore blocks translocation of notch intracellular domain to the nucleus. "
    [Show abstract] [Hide abstract] ABSTRACT: Pollen allergens are delivered to epithelial surfaces of the upper respiratory tract in conjunction with multiple endogenous adjuvants. We previously demonstrated pollen-mediated modulation of cytokine and chemokine production of dendritic cells, contributing to a Th2-dominated micromilieu. As T helper cell differentiation not only depends on dendritic cell-derived cytokines but also on cell-cell-contact mediated mechanisms, we studied the expression of notch ligands and myeloid differentiation primary response protein 88 (MyD88) in dendritic cells matured in the presence of aqueous birch pollen extracts and pollen-associated E1-phytoprostanes. Human monocyte-derived dendritic cells were stimulated with aqueous birch pollen extracts in the absence or presence of lipopolysaccharide, and mRNA expression levels of notch ligands delta-1 and -4, jagged-1 and -2 and of myd88 were determined. Regulation of Delta-4 and MyD88 by aqueous pollen extracts was assessed on protein level. The contribution of notch signaling to T helper cell differentiation was analyzed in allogeneic T cell stimulation assays. In immature dendritic cells, stimulation with pollen extracts resulted in an induction of both delta and jagged notch ligands. The lipopolysaccharide-induced up-regulation of delta-1 and -4 and of myd88 was decreased by aqueous pollen extracts, whereas jagged expression was induced. Reduction of Delta-4 and MyD88 by aqueous pollen extracts was confirmed on protein level. The Th2-skewing activity was contained in a fraction of aqueous pollen extracts enriched for molecules <3 kDa and was distinct from the previously identified E1-phytoprostanes. Reduction of notch signaling in dendritic cells matured in the presence aqueous pollen extract leads to inhibition of IL-10 and to induction of IL-5 production in naïve T cells differentiated by these dendritic cells. Pollen derived, non-allergenic factors reduce the dendritic cell's expression of Th1 instructing Delta-like notch ligands and of MyD88, thereby promoting Th2 skewing of T helper cell responses.
    Full-text · Article · Dec 2015 · World Allergy Organization Journal
    • "However, closer inspection revealed that Th1 cell differentiation per se was unaffected in these mice. Inability to clear the infection was in fact traced to defective secretion of IFNg, while the number of Th1 cells (identified by intracellular expression of IFNg and T-bet) was normal [17]. By sharp contrast, Th1 responses and resistance to L. major were unaffected in mice expressing a dominant negative MAML (DN MAML) transgene, which did nonetheless effectively inhibit canonical Notch signaling [24] . "
    [Show abstract] [Hide abstract] ABSTRACT: Differentiation of naïve T cells into effector cells is required for optimal protection against different classes of microbial pathogen and for the development of immune memory. Recent findings have revealed important roles for the Notch signaling pathway in T cell differentiation into all known effector subsets, raising the question of how this pathway controls such diverse differentiation programs. Studies in preclinical models support the therapeutic potential of manipulating the Notch pathway to alleviate immune pathology, highlighting the importance of understanding the mechanisms through which Notch regulates T cell differentiation and function. We review these findings here, and outline both unifying principles involved in Notch-mediated T cell fate decisions and cell type- and context-specific differences that may present the most suitable points for therapeutic intervention.
    No preview · Article · Nov 2015 · Trends in Immunology
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    • "Mice lacking notch signaling in CD4+ T cells were shown to have defective Th2 cell differentiation, but they were able to differentiate in Th1 cells and control Leishmania major infection [15]. Also, Notch receptors were shown to be necessary for gamma interferon production by murine Th1 cells during Leishmania major infection [16] . Interestingly , the positional associations on chromosomes 1 and 2 found in the present study point to the same resistance mechanism, which involves parasite killing triggered by T helper 1 cells and macrophage signaling. "
    [Show abstract] [Hide abstract] ABSTRACT: We conducted a genome-wide scan for visceral leishmaniasis in mixed-breed dogs from a highly endemic area in Brazil using 149,648 single nucleotide polymorphism (SNP) markers genotyped in 20 cases and 28 controls. Using a mixed model approach, we found two candidate loci on canine autosomes 1 and 2. The positional association on chromosome 2 mapped to a predicted DNAse sensitive site in CD14+ monocytes that serve as a cis-regulatory element for the expression of interleukin alpha receptors 2 (IL2RA) and 15 (IL15RA). Both interleukins were previously found to lead to protective T helper 1 cell (Th1) response against Leishmania spp. in humans and mice. The associated marker on chromosome 1 was located between two predicted transcription factor binding sites regulating the expression of the transducin-like enhancer of split 1 gene (TLE1), an important player in Notch signaling. This pathway is critical for macrophage activity and CD4+ T cell differentiation into Th1 and T helper 2. Together, these findings suggest that the human and mouse model for protective response against Leishmania spp., which involves Th1 and macrophage modulation by interleukins 2, 15, gamma interferon and Notch signaling, may also hold for the canine model.
    Full-text · Article · Sep 2015 · PLoS ONE
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