Altered Dendritic Cell Phenotype in Response to Leishmania amazonensis Amastigote Infection Is Mediated by MAP Kinase, ERK

Article (PDF Available)inAmerican Journal Of Pathology 174(5):1818-26 · May 2009with24 Reads
DOI: 10.2353/ajpath.2009.080905 · Source: PubMed
Abstract
Initiation of productive immune responses against Leishmania depends on the successful transition of dendritic cells (DC) from an immature to a mature phenotype. This process is characterized by high CD40 surface expression as well as interleukin-12 production, which are frequently seen in response to L. major infection. In vivo footpad infection of C3HeB/FeJ mice for 7 days with L. amazonensis promoted an immature CD11c(+) DC phenotype characterized by both significantly low CD40 surface expression and significantly decreased interleukin-12p40 production compared with L. major infection of these same mice. In vitro infection of bone marrow-derived dendritic cells with L. amazonensis amastigotes resulted in rapid and significant phosphorylation of the mitogen activated protein kinase, extracellular signal-regulated kinase 1/2, observed within minutes of exposure to the parasite. Infection with L. amazonensis promastigotes led to increased 1/2 phosphorylation after 4 hours of infection compared with L. major infection, which correlated with promastigote transformation into amastigotes. Treatment of bone marrow-derived dendritic cells with a mitogen activated protein kinase kinase-specific inhibitor, PD98059, led to regained surface CD40 expression and interleukin-12p40 production following L. amazonensis amastigote infection compared with non-treated, infected DC. Treatment of L. amazonensis-infected mice with the highly-specific mitogen activated protein kinase kinase inhibitor, CI-1040, enhanced surface CD40 expression on CD11c(+) DC obtained from the draining lymph node. L. amazonensis amastigotes, through activation of extracellular signal-regulated kinase 1/2, inhibit the ability of DC to undergo proper maturation both in vitro and in vivo.
Immunopathology and Infectious Diseases
Altered Dendritic Cell Phenotype in Response to
Leishmania amazonensis Amastigote Infection Is
Mediated by MAP Kinase, ERK
Paola Mercedes Boggiatto,*
Fei Jie,* Mousumi Ghosh,
†‡
Katherine Nicole Gibson-Corley,*
Amanda Ellen Ramer-Tait,
§
Douglas Elliot Jones,*
and Christine Anne Petersen*
From the Immunobiology Program,* Department of Veterinary
Pathology,
and Department of Veterinary Microbiology and
Preventive Medicine,
§
College of Veterinary Medicine Iowa State
University, Ames, Iowa; and the Lois Pope LIFE Center,
University of Miami Leonard M. Miller School of Medicine,
Miami, Florida
Initiation of productive immune responses against
Leishmania depends on the successful transition of
dendritic cells (DC) from an immature to a mature
phenotype. This process is characterized by high
CD40 surface expression as well as interleukin-12
production, which are frequently seen in response to
L. major infection. In vivo footpad infection of
C3HeB/FeJ mice for 7 days with L. amazonensis pro-
moted an immature CD11c
DC phenotype character-
ized by both significantly low CD40 surface expres-
sion and significantly decreased interleukin-12p40
production compared with L. major infection of these
same mice. In vitro infection of bone marrow-derived
dendritic cells with L. amazonensis amastigotes re-
sulted in rapid and significant phosphorylation of the
mitogen activated protein kinase, extracellular sig-
nal-regulated kinase 1/2, observed within minutes of
exposure to the parasite. Infection with L. amazonen-
sis promastigotes led to increased 1/2 phosphoryla-
tion after 4 hours of infection compared with L. major
infection, which correlated with promastigote trans-
formation into amastigotes. Treatment of bone mar-
row-derived dendritic cells with a mitogen activated
protein kinase kinase-specific inhibitor, PD98059,
led to regained surface CD40 expression and interleu-
kin-12p40 production following L. amazonensis amas-
tigote infection compared with non-treated , infected
DC. Treatment of L. amazonensis-infected mice with the
highly-specific mitogen activated protein kinase kinase
inhibitor, CI-1040 , enhanced surface CD40 expression
on CD11c
DC obtained from the draining lymph node.
L. amazonensis amastigotes , through activation of ex-
tracellular signal-regulated kinase 1/2 , inhibit the abil-
ity of DC to undergo proper maturation both in vitro
and in vivo.
(Am J Pathol 2009, 174:1818 –1826; DOI:
10.2353/ajpath.2009.080905)
Leishmaniasis is a vector-borne disease caused by intra-
cellular protozoan parasites of the genus Leishmania. The
intracellular amastigote stage of Leishmania predomi-
nates in the mammalian host while the vector stage exists
as an extracellular, flagellated promastigote. Both pro-
mastigotes and amastigotes are capable of initiating
mammalian infection. Leishmania amastigotes have been
shown to interfere with host cell function, including mod-
ulation of signaling pathways, suppression of antimicro-
bial and pro-inflammatory mediators, and induction of
cytokines that promote disease progression.
1,2
Infection of C3HeB/FeJ mice with Leishmania major
leads to the development of a healing T
H
1 immune re-
sponse characterized by high levels of interleukin-12 and
interferon (IFN)-
-producing CD4
T cells.
3
In contrast,
infection with L. amazonensis results in a non-healing
immune response, leading to chronic disease and high
parasite loads. Multiple studies have investigated and
identified differences in the T cell response to these two
parasites.
4–6
Studies from our laboratory have revealed
that antigen-responsive CD4
T cells from C3HeB/FeJ
mice chronically infected with L. amazonensis are im-
paired in their ability to transition from a naïve to an
effector phenotype.
6
Leishmania spp. infect phagocytic cells of the immune
system, primarily macrophages and dendritic cells (DC).
DC are antigen presenting cells that efficiently initiate
Supported by National Institutes of Health grants AI48357, AI50803.
Accepted for publication January 27, 2009.
Address reprint requests to Christine Petersen, Department of Veteri-
nary Pathology, Iowa State University, 1600 University Boulevard, Ames,
IA 50011. E-mail: kalicat@iastate.edu.
The American Journal of Pathology, Vol. 174, No. 5, May 2009
Copyright © American Society for Investigative Pathology
DOI: 10.2353/ajpath.2009.080905
1818
antigen-specific immune responses by inducing the dif-
ferentiation of naïve T cells.
7
DC maturation can be char
-
acterized in vitro and ex vivo by the upregulation of co-
stimulatory molecules including CD40 and the production
of T cell-polarizing cytokines such as interleukin (IL)-12.
7
Clearance and resistance to Leishmania infection is de-
pendent on the development of a T
H
1 response
3,8
and
susceptibility to Leishmania infection has been associ-
ated with deficiencies in CD40, CD40 ligand,
9 –11
and
IL-12 or IL-12 receptor subunits.
12,13
Infection of C57BL/6
mice bone marrow-derived dendritic cells (BMDC) with L.
amazonensis promastigotes indicated a significant reduc-
tion in CD40 surface expression and IL-12p40 production
as compared with L. major promastigote-infected BMDC.
14
Moreover, a recent publication indicated that L. ama-
zonensis amastigote infection of BMDC significantly
down-regulated CD40 expression and suppressed IL-
12p40 and IL-12p70 production, leading to an impair-
ment in antigen presenting cell function, characterized by
an inability of L. amazonensis-amastigote infected BMDC
to prime naïve CD4
T cells or re-stimulate antigen-
specific CD4
T cells.
15
Improper or insufficient activation of DC can promote a
non-polarized, often tolerogenic, T cell response. DC
maturation depends on the balance of particular molec-
ular signaling cascades.
7,16,17
One such pathway is
mitogen activated protein kinase (MAPK) signaling
cascades composed of three primary kinases, p38,
c-Jun N-terminal kinase (JNK), and extracellular sig-
nal-regulated kinase (ERK). A recent report has linked
ERK activation in macrophages with the induction of
IL-10, a critical cytokine involved in host susceptibility
to Leishmania infection.
18
Work from our own laboratory
has shown that inhibition of ERK in vitro increases the
ability of L. amazonensis-infected macrophages to kill
the intracellular parasites.
19
Unlike p38 and JNK, ERK
has been shown to play a role in preventing proper
maturation of DC.
16,20
As L. amazonensis-infected mice fail to develop an
effective T
H
1 response,
4–6
and L. amazonensis-infected
BMDC in vitro have been shown to have an altered mat-
uration phenotype,
14,15
we hypothesized that L. ama
-
zonensis infection in vitro promotes an immature DC phe-
notype consistent with the observed lack in T cell
polarization. In this study, we present novel ex vivo evi-
dence that L. amazonensis infection promotes an imma-
ture CD11c
DC phenotype characterized by signifi
-
cantly low CD40 surface expression and significantly
decreased IL-12p40 production as compared with L. ma-
jor infection. Furthermore, we explored the molecular
mechanisms that may lead to impaired DC maturation
and found that in vitro, BMDC infection with L. amazonen-
sis amastigotes resulted in rapid and significant phos-
phorylation of the MAP kinase ERK1/2, observed within
minutes of exposure to the parasite. Infection with L.
amazonensis promastigotes led to increased ERK1/2
phosphorylation as compared with L. major infection;
however, this phosphorylation was delayed several
hours. This delay in phosphorylation correlated with pro-
mastigote transformation into amastigotes within infected
DC, as confirmed by microscopic analysis of parasite
stage before and after initiation of robust ERK phosphor-
ylation. In vitro inhibition studies determined that treat-
ment of DC with a mitogen activated protein kinase ki-
nase (MEK)-specific inhibitor, PD98059, led to enhanced
surface CD40 expression and IL-12p40 production fol-
lowing L. amazonensis-amastigote infection as compared
with non-treated cells. Treatment of L. amazonensis-in-
fected mice with the highly-specific MEK inhibitor, CI-
1040, enhanced surface CD40 expression. Together,
these data indicate that L. amazonensis amastigotes,
through activation of the MAP kinase ERK1/2, inhibit the
ability of DC to undergo proper maturation in vivo. This is
the first report of use of a biochemical inhibitor, here
targeted to the MAPK ERK, which restores the immune
phenotype of dendritic cells after pathogen infection both
in vitro and in vivo.
Materials and Methods
Mice
C3HeB/FeJ mice were bred in house or obtained from
Jackson Laboratories (Bar Harbor, ME) and maintained
in a specific-pathogen-free facility. The Institutional Ani-
mal Care and Use Committee at Iowa State University
approved all protocols involving animals. Six- to eight-
week old females were inoculated with 1 10
6
station
-
ary-phase promastigotes in 50
l of PBS in the left hind
footpad.
C3H severe combined immunodeficiency mice (C3SnSmn.
CB17-Prkdc
scid
/J) were inoculated with 1 to 2 10
7
stationary-phase promastigotes in 50
l of PBS in the left
hind footpad. These mice were later sacrificed for tissue-
derived amastigotes.
For in vivo ERK inhibitor treatment, L. amazonensis- and
L. major-infected C3HeB/FeJ mice were orally gavaged
twice daily with 100 mg/kg of CI-1040 (kind gift from
Pfizer Global Health, Groton, CT) in dimethyl sulfoxide
supplemented with 0.5% hydroxypropyl methyl cellulose
and 0.2% Tween 80, for a total of 7 days and sacrificed.
Isolation and Preparation of BMDC
BMDC were cultured in vitro in the presence of 10 ng/ml
of murine granulocyte-macrophage colony-stimulating
factor (PeproTech Inc., Rocky Hill, NJ) according to the
method of Lutz et al.
21
At day 10 of culture, approximately
90% of the BMDC were positive for the DC marker
CD11c. For in vitro studies, 10-day-old BMDC were incu-
bated for 24 hours at 37°C, 5% CO
2
with fresh, tissue-
derived amastigotes at a cell to parasite ratio of 1:3.
Parasites and Infection
Culture of L. amazonensis (MHOM/BR/00/LTB0016) and
L. major (MHOM/IL/80/Friedlin) parasites was performed
as previously described.
5
For in vitro promastigote exper
-
iments, stationary phase L. amazonensis or L. major pro-
mastigotes were used. Where indicated, parasites were
labeled using the dye carboxyfluorescein diacetate suc-
L. amazonensis Alters DC Phenotype 1819
AJP May 2009, Vol. 174, No. 5
cinimidyl ester (Molecular Probes, Eugene, OR) as pre-
viously described
22
with some modifications. Amasti
-
gotes were directly recovered from footpad lesions of
infected C3H severe combined immunodeficiency mice.
Infected feet were disinfected with 70% ethanol, and
extraneous tissue dissected away. The remaining lesion
was then homogenized with a Tenbrock tissue homoge-
nizer. Cellular debris was removed by centrifuging at
180 g for 15 minutes at 4°C. The resulting supernatant
was centrifuged at 3000 g for 15 minutes at 4°C. The
number of viable amastigotes in the pellet was assessed
by fluorescent microscopy with fluorescein diacetate (Ac-
ros Organics, Morris Plains, NJ) and propidium iodide
(Sigma, St. Louis, MO). For in vitro infection of BMDC,
cells were infected with either L. amazonensis or L. major
at a multiplicity of infection of 3. Where indicated, as a
positive control for maturation, some BMDC were stimu-
lated with 500 ng of lipopolysaccharide (Sigma, St. Louis,
MO) and 200iU of IFN-
(Pharmigen, San Diego, CA), or
treated with the MEK inhibitor PD98059 (Sigma, St.
Louis, MO).
Flow Cytometry
For flow cytometry analysis of surface molecule expres-
sion, 1 10
6
BMDC were washed in 2 ml of fluores
-
cence-activated cell sorting buffer (FACS, 0.1% sodium
azide and 0.1% bovine serum albumin in phosphate
buffer saline). Fc
receptors were blocked with 10% pu-
rified rat anti-mouse CD16/CD32 antibody (BD Pharmin-
gen, San Diego, CA) in 1 mg/ml rat IgG for 20 minutes at
4°C to prevent nonspecific binding. BMDC were then
incubated with the appropriate antibody or isotype con-
trol for 30 minutes on ice. The antibodies used include
fluorescein isothiocyanate-labeled CD11c (HL3), PerCP-
Cy5.5-labeled CD11b (M1/70), phycoerythrin-labeled
CD40 (3/23), allophycocyanin-labeled CD80 (16-10A1),
phycoerythrin-Cy7-labeled CD86 (GL-1), allophycocya-
nin-Cy7-labeled CD19 (1D3), and allophycocyanin-Cy7-
labeled CD3e (145-2C11). CD11c, CD11b, CD40, CD19,
and CD3 were purchased from BD Pharmigen (San
Diego, CA). CD80 and CD86 were purchased from
Biolegend (San Diego, CA). Following staining, cells were
washed in 2 ml of FACS buffer and fixed in 200
lof1%
paraformaldehyde and stored at 4°C until analysis. Anal-
ysis was performed on a BD FACScanto flow cytometer
(Becton Dickinson, San Jose, CA), and data anal-
yzed using FlowJo software V8.5.2 (Tree Star, Inc.,
Ashland, OR).
Isolation of Cell Extracts
To make whole cell lysates, 3 10
6
BMDC were resus
-
pended in 400
lof1 cell lysis buffer (Cell Signaling
Technologies, Beverly, MA), supplemented with 1
mmol/L phenylmethylsulphonyl fluoride and a protease
inhibitor cocktail (Roche, Indianapolis, IN) immediately
before use. Samples were incubated on ice for 15 min-
utes and then centrifuged at 16,000 g for 5 minutes at
4°C. Supernantants were collected as whole cell lysates
and stored at 80°C.
Immunoblot Analysis
Protein content of all cell extracts was determined via
BCA protein assay (Pierce, Rockford, IL) according to
manufacturer’s recommendations, and all samples were
normalized to 1 mg/ml using distilled water. Samples (20
to 30
g of protein) were heated for 4 minutes at 95°C in
1 loading buffer and electrophoresis was performed on
a 12% SDS-polyacrylamide electrophoresis gel. Gels
were electroblotted onto polyvinylidene fluoride mem-
branes, blocked with 5% bovine serum albumin, and
probed with antibodies specific for phospho-ERK, p38
and JNK, total-ERK1/2, p38, JNK (1:1000) (Cell Signal-
ing, Beverly, MA), and
-actin (1:5000) (Sigma, St. Louis,
MO). Signals were detected with horseradish-peroxi-
dase-conjugated goat anti-rabbit antibodies (1:20,000)
(Jackson ImmunoResearch, West Grove, PA) using the
SuperSignal West chemiluminescent substrate (Pierce,
Rockford, IL) and expressed to autoradiography film
(Midsci, St. Louis, MO).
Indirect Immunofluorescence and H&E Stain
BMDC (5 10
5
) were plated onto 24-well plates contain
-
ing tissue coverslips. BMDC were infected with L. ama-
zonensis promastigotes at a multiplicity of infection of 3:1
and incubated at 34°C with 5% CO
2
. Tissue coverslips
were then harvested and fixed with 4% paraformalde-
hyde in PBS for 20 minutes at room temperature and
washed three times with PBS. BMDC were permeabilized
with 0.1% saponin in PBS for 10 minutes at room temper-
ature. Cells were incubated for 1 hour at room tempera-
ture with mouse anti-LPG CA7AE monoclonal antibody
(kind gift from Dr. Jeffery Beetham, Iowa State University)
at a 1:100 dilution in 0.1% saponin. After incubation,
coverslips were washed three times with PBS and incu-
bated for 1 hour at room temperature with rabbit anti-
mouse Cy2-conjugated antibody (Jackson Immuno-
Research Laboratories, West Grove, PA, kind gift from
Dr. Bryan Bellaire, Iowa State University) at a 1:200
dilution in 0.1% saponin. BMDC were then counter-
stained with propidium iodide according to manufac-
turer’s instructions (Molecular Probes, Eugene, OR).
Coverslips were then mounted onto slides using
MOWIOL (Calbiochem, La Jolla, CA) and viewed using
an Olympus IX71 inverted epifluorescence scope
(Olympus America Inc., Center Valley, PA); 300 in-
fected cells were counted on each coverslip.
For morphological analysis, coverslips were harvested
at the indicated time points, fixed in 100% methanol for 5
minutes and stained with H&E according to manufactur-
er’s instructions (Fisher Diagnostics, Middletown, VA).
Coverslips were analyzed using a Nikon Eclipse 50i light
microscope (Nikon Instruments Inc., Melville, NY).
1820 Boggiatto et al
AJP May 2009, Vol. 174, No. 5
IL-12p40 Enzyme-Linked Immunosorbent Assay
and ELIspot
Supernatants from BMDC cultures were harvested at indi-
cated time points following amastigote or promastigote in-
fection, and IL12-p40 enzyme-linked immunosorbent assay
was performed using commercially available antibodies
(BD Pharmigen, San Diego, CA), peroxidase-conjugated
streptavidin (Jackson ImmunoResearch Laboratories, West
Grove, PA) and ABTS microwell peroxidase substrate
(Roche, Indianapolis, IN). IL-12p40 ELIspots were per-
formed on total lymph node cells using commercially avail-
able purified IL-12p40 and biotinylated anti-IL12p40 anti-
bodies (BD Pharmigen, San Diego, CA), and developed
using 2-amino-2-methyl-1-propanol (ICN Biomedicals Inc.,
Aurora, OH) and 5-bromo-4-chloro-3-indoly-phosphate
(Fisher, Fair Lawn, NJ).
Statistics
Statistical analysis between two values was determined
via Student’s t-tests. P values of 0.05 were considered
as statistically significant.
Results
DC in the Draining Lymph Node of
L. amazonensis-Infected Mice Have an
Immature Phenotype
Recent studies demonstrated that BMDC infected with L.
amazonensis amastigotes have an immature phenotype,
as determined by CD40 expression and IL-12p40 and
IL-12p70 production, and are impaired in their ability to
prime naïve CD4
T cells as compared with L. amazonen
-
sis promastigote-infected BMDC.
15
Our laboratory has
shown that CD4
T cells from mice chronically infected
with L. amazonensis are impaired in their ability to transi-
tion into an effector phenotype.
6
We propose that these
differences in BMDC phenotype previously observed to
be elicited by L. amazonensis promastigotes and amasti-
gotes in vitro, would be reflected during in vivo infection as
the infection progresses from peracute (2 days post-
infection [dpi]) to acute (7 dpi). Mice were infected sub-
cutaneously in the left footpad with L. amazonensis or L.
major promastigotes and sacrificed at 2 and 7 dpi. DC, as
designated by CD11c
cells
23
from the draining lymph
node (DLN) of infected mice, collected based on CD19
and CD3 double negative events (Figure 1A), were ana-
lyzed for DC maturation markers. As shown previously,
infection with Leishmania not only increases the number
of CD11c
-events in the DLN, but also increases CD40
expression in these cells.
23
CD11c
DC surface expres
-
sion of CD40 (Figure 1, B and D), CD80 and CD86 (data
not shown) was similar between L. amazonensis-infected
and L. major-infected mice at 2 dpi. By 7 dpi, CD11c
DC
from L. amazonensis-infected mice had significantly lower
CD40 surface expression as measured by FACS analysis
as compared with DC from L. major-infected mice (Figure
1, C and E). No differences were observed in the surface
expression of CD80 and CD86 (data not shown) between
L. amazonensis-infected and L. major-infected mice at
7dpi, suggesting that CD40 expression is specifically
targeted for down-regulation.
Figure 1. Decreased CD40 expression and IL-
12p40-producing cells from DC subsets of total
draining lymph node cells from L. amazonensis-
infected mice at 7 days postinfection. Mice were
infected with L. amazonensis or L. major promas-
tigotes and sacrificed at 2 and 7 days post-infec-
tion. CD11c
DC subsets from total draining
lymph node cells, collected on CD19 and CD3
double negative events (A) from L. amazonensis-
infected, L. major-infected, and naïve mice. Cells
were analyzed for CD40 surface expression via
FACS scan. Histograms (B and C) indicate percent
CD40 positive CD11c
CD11b
cells (gray, isotype
control; black, CD40) and bar graphs (D and E)
indicate CD40 mean fluorescence intensity of cells
at 2dpi (B and D) and 7 dpi (C and E). F and G:
Number of IL-12-producing cells from DLN of mice
infected with L. amazonensis or L. major at 2dpi
(F)or7dpi(G) measured via ELispot. All data are
from three separate experiments; error bars indi-
cate SEM; *P 0.05.
L. amazonensis Alters DC Phenotype 1821
AJP May 2009, Vol. 174, No. 5
The number of DLN cells producing IL-12p40 ex vivo
was analyzed at 2 and 7 dpi via ELIspot. At 2 dpi, no
significant differences were observed in IL-12p40 pro-
duction from the DLN of L. amazonensis- or L. major-
infected mice (Figure 1F). However, by 7 dpi the number
of IL-12p40-producing cells was significantly decreased
in response to L. amazonensis infection compared with L.
major-infected mice (Figure 1G). These data suggest that
early during infection, L. amazonensis and L. major pro-
mastigotes elicit similar DC phenotypes in the DLN, as
measured by CD40 surface expression and IL-12p40
production. However, once the in vivo infection transitions
to the acute stage (7 dpi) when the amastigote form of the
parasite predominates,
24
we observe a DC phenotype
with significantly decreased CD40 surface expression
and IL-12p40 production suggesting that L. amazonensis
amastigotes can modulate the maturation phenotype of
DC in vivo as it has been observed in vitro.
Increased ERK Phosphorylation Following
Infection with L. amazonensis Amastigotes
To determine a potential mechanism by which L. ama-
zonensis could inhibit CD40 surface expression and IL-12
production during DC maturation, we determined which
of several signaling pathways are differentially up-regu-
lated in DC following infection with L. amazonensis and
not with L. major infection. Several reports indicate that
activation of mitogen-activated protein kinase ERK can
prevent proper DC maturation.
16,20
Recent published
work from our laboratory and others has shown that ac-
tivation of ERK1/2 following infection with L. amazonensis
amastigotes leads to decreased ability of mouse macro-
phages to kill these intracellular parasites in vitro,
19
and
promotes a non-healing response in infected BALB/c
mice.
18
Based on these findings, we were interested in
determining whether ERK phosphorylation occurs in
C3HeB/FeJ-derived BMDC after infection with either L.
amazonensis amastigotes or promastigotes. BMDC in-
fected with L. amazonensis amastigotes (Figure 2A) or
promastigotes (Figure 2, B and C) at a 3:1 parasite-cell
ratio were lysed at the indicated time points and analyzed
via Western blot for phosphorylated ERK1/2. Following L.
amazonensis amastigote infection, a significant increase
in ERK1/2 phosphorylation was observed within minutes
of exposure to the parasite (Figure 2A), compared with L.
major amastigote infection. In contrast, L. amazonensis
promastigote infection did not result in significantly in-
creased phosphorylated ERK1/2 until at least 3 hours
post-infection (Figure 2, B and C) as compared with L.
major promastigote-infected BMDC. Phosphorylation of
ERK1/2 after L. amazonensis amastigote infection was
Figure 2. Robust ERK1/2 phosphorylation in L. amazonensis amastigote-infected BMDC and delayed ERK1/2 activation following L. amazonensis promastigote
infection. BMDC were infected with L. amazonensis or L. major amastigotes (A) or promastigotes (B and C). Whole cell lysates were collected at the indicated
time points. Samples were analyzed via Western blot for phosphorylated ERK1/2 (A–C), p38 (D), JNK (E) and total ERK1/2, p38 and JNK. Densitometry values
were normalized to t-ERK, t-p38, or t-JNK and then to noninfected controls. Densitometry analysis for at least three different experiments and representative blots
are shown; error bars denote SD; *P 0.05.
1822 Boggiatto et al
AJP May 2009, Vol. 174, No. 5
unique to ERK1/2 as other MAP kinases, p38 and JNK, were
not up-regulated following infection (Figure 2, D and E).
We hypothesized that the observed delay in ERK1/2
phosphorylation following L. amazonensis promastigote
infection may correlate with intracellular transformation of
the parasite into the amastigote stage. We analyzed L.
amazonensis-infected BMDC at 2 and 4 hours postinfec-
tion to determine which parasite form, promastigote or
amastigote, predominated at these time points in infected
cells. Using both immunofluorescence via an anti-LPG
antibody (Figure 3A), which selectively labels promastig-
otes and morphology analysis via H&E stain (Figure 3B),
we show that while at 2 hours postinfection parasite form
is dominated by promastigotes, the percentage of pro-
mastigotes present intracellularly is significantly de-
creased by 4 hours postinfection, indicating a growing
predominance of amastigote infection. Four hours postin-
fection correlates with the rise of increased ERK1/2 phos-
phorylation observed after initial promastigote infection.
These data suggest that L. amazonensis amastigotes may
specifically induce ERK1/2 phosphorylation.
ERK Inhibition Restores CD40 Surface
Expression Both in Vitro and in Vivo Following
L. amazonensis Infection
We have shown that MAPK ERK1/2 is selectively and
rapidly phosphorylated following L. amazonensis amasti-
gote infection of BMDC. To examine if increased ERK1/2
phosphorylation affects BMDC maturation in response to
activating stimuli following Leishmania infection, BMDC
were pre-treated with the specific MEK inhibitor PD98059
and then infected with either L. amazonensis or L. major
amastigotes and activated with lipopolysaccharide and
IFN-
2 hours postinfection. Following overnight culture,
BMDC were analyzed for CD40 surface expression via
FACS and resultant culture supernatants were analyzed
to determine IL-12p40 production via enzyme-linked im-
munosorbent assay. ERK inhibition of L. amazonensis
amastigote-infected BMDC significantly increased CD40
surface expression MFI (Figure 4A) and IL-12p40 pro-
duction (Figure 4B) as compared with non-treated L.
amazonensis amastigote-infected BMDC. Treatment with
PD98059 had no effect on the ability of L. major-infected
BMDC to express surface CD40 as measured by FACS
analysis or to produce IL-12p40 (data not shown).
Based on these in vitro findings, where inhibition of
ERK before L. amazonensis amastigote infection of BMDC
enhanced CD40 expression and IL-12p40 production,
we sought to determine the effect of ERK inhibition in vivo
using a different ERK inhibitor, CI-1040. Mice were inoc-
ulated in the left footpad with stationary phase L. ama-
zonensis or L. major promastigotes. Starting day 0 mice
were treated with CI-1040 via oral gavage. Twice daily
treatment continued for a total of 7 days. Mice were
sacrificed 1 week after infection, DLNs were harvested,
and CD11c
DC phenotypes were analyzed via FACS as
described previously (Figure 1). ERK inhibitor treatment
of L. amazonensis-infected mice resulted in increased
CD11c
DC surface expression of CD40 (Figure 4C [left
panel] and D) as determined by FACS analysis. ERK
inhibitor treatment significantly decreased the number of
IL-12p40-producing cells as measured by ELIspot (Fig-
ure 4F). CI-1040 treatment of L. major-infected mice had
no effect on CD40 surface expression of CD11c
DC
(Figure 4C [right panel] and E) or on the number of
IL-12p40 producing cells from the DLNs of infected mice
(Figure 4G). Western blot analysis of splenic lysates
showed that ERK1/2 phosphorylation was inhibited in
CI-1040-treated animals (Figure 4H). These data indicate
that in vitro L. amazonensis infection modulates DC matu-
ration via ERK-mediated down-regulation of CD40 sur-
face expression and decreased IL-12p40 production,
and that in vivo, ERK activation down-regulates CD40
surface expression of CD11c
DC.
Discussion
Here we describe a novel L. amazonensis amastigote-
dependent mechanism modulating DC maturation via ac-
tivation of the MAP kinase ERK and for the first time
recover DC phenotype by inhibiting ERK phosphoryla-
tion. Alteration of surface marker expression, cytokine
production, maturation, and function of DC following L.
amazonensis infection has been previously characterized
in vitro.
15,25
Defects in DC maturation may, in part, con
-
tribute to the unpolarized T cell phenotype observed
during L. amazonensis infection,
6
and lead to a non-heal
-
ing immune response. As early as 7 dpi CD11c
DLN
cells from L. amazonensis-infected C3HeB/FeJ mice have
significantly reduced CD40 surface expression and a
decreased number of IL-12p40-producing cells, as com-
pared with L. major-infected mice. Consistent with an-
other report indicating the activation of the MAP kinase
ERK during L. amazonensis infection,
18
we found that L.
amazonensis amastigote infection of DC leads to phos-
phorylation of the MAP kinase ERK1/2. When ERK phos-
phorylation was inhibited, DC surface expression of
CD40 increased both in vitro and in vivo, and production
of IL-12p40 was enhanced in vitro.
Figure 3. L. amazonensis amastigotes predominate within infected cells at
4 hours post-infection. BMDC in 24 well plates containing coverslips were
infected with L. amazonensis promastigotes. Coverslips were recovered at
the indicated time points, fixed, and stained. A: Epifluorescent microscopy
analysis of BMDC, (100, oil). Infected cells were determined by propidium
iodide (PI) nuclear staining, and promastigotes were identified via a CA7AE
anti-LPG antibody and a secondary Cy2-conjugated antibody. Graph indi-
cates mean and SD of 3 coverslips of one representative from two experi-
ments. B: Coverslips were analyzed via light microscopy, (100, oil). Parasite
stage was identified by presence or absence of flagellum and by size. Data
from at least three separate experiments; error bars denote SEM; *P 0.05.
L. amazonensis Alters DC Phenotype 1823
AJP May 2009, Vol. 174, No. 5
Several pathways that alter DC maturation have been
characterized, including MAP kinase pathways.
16,20
p38
and JNK phosphorylation were not different between L.
amazonensis-infected and L. major-infected BMDC (Fig-
ure 2, D and E). ERK1/2 phosphorylation in BMDC in-
creased fourfold within minutes of contact with L. ama-
zonensis amastigotes compared with L. major amastigotes
(Figure 2A). ERK1/2 phosphorylation was also observed
after L. amazonensis promastigote infection of BMDC but not
until 3 to 4 hours post-infection (Figure 2C). Early ERK1/2
phosphorylation observed with in vitro following L. ama-
zonensis amastigote infection of BMDC (Figure 2A) and
microscopic analysis revealing that the amastigote form of
L. amazonensis predominates within infected BMDC by 4
hours post-infection (Figure 3), suggest a correlation be-
tween ERK1/2 phosphorylation and amastigote predomi-
nance. We postulate that the amastigote form of L. ama-
zonensis activates the ERK1/2 pathway.
ERK1/2 has multiple cytosolic and nuclear targets.
26,27
The role of ERK1/2 in cellular function ranges from cell
survival and cell cycle regulation, to modulation of addi-
tional signaling pathways and regulation of transcription.
Both CD40 and IL-12p40 transcription can be negatively
regulated by activation of ERK1/2.
20
ERK1/2 has also
been demonstrated to play a role in the DC survival rather
than maturation.
16
Moreover, in tumor cells, aberrant ac
-
tivation of ERK1/2 is implicated in inhibition of differenti-
ation and apoptosis.
28
Activation of ERK1/2 in infected
DC by L. amazonensis amastigotes may provide two crit-
ical mechanisms to ensure parasite success within the
mammalian host. First, by targeting specific genes for
regulation, namely CD40 and IL-12p40, L. amazonensis
could interfere with proper DC maturation, thereby pre-
venting immune detection and induction of a proper
adaptive immune response. Second, increased activa-
tion of host ERK1/2 would promote survival of the host cell
therefore maintaining a viable host cell for an extended
period of time.
ERK1/2 activation as an immunomodulatory mecha-
nism for leishmaniasis has been previously described in
other cells systems. Leishmania phosphoglycan has been
shown to inhibit IL-12 production by macrophages via
ERK1/2 activation.
29
Recent work has demonstrated that
antibody-opsonized L. amazonensis amastigotes induce
ERK1/2 activation in BALB/c macrophages. Our work
using a C3HeB/FeJ mouse model complement these
findings, providing further support for ERK in the patho-
genesis of L. amazonensis infection. We demonstrate that
Figure 4. ERK inhibition enhances DC matura-
tion phenotype following L. amazonensis infec-
tion (A–B) BMDC were pretreated with the ERK
inhibitor PD98059 (20 mmol/L) for 30 minutes and
then infected with L. amazonensis amastigotes,
and activated with LPS and IFN-
. 24 hours post-
infection cells were harvested and processed for
(A) surface CD40 expression and analyzed via
FACS scan and (B) supernatants were collected to
determine IL-12p40 production via enzyme-linked
immunosorbent assay. C–H: Mice were infected
with L. amazonensis or L. major promastigotes
and then treated with 100 mg/kg of CI-1040 via
oral gavage started on day 0 post-infection and for
the next 7 days, twice daily. On day 7 mice were
sacrificed; draining lymph nodes were harvested
to assess CD40 surface expression on DC popula-
tions. C: Representative histograms based on
CD11c
CD11b
population, (gray line, isotype
control; black CD40). Mean fluorescence intensity
of CD40 surface expression on CD11c
cells in L.
amazonensis-infected (D)orL. major-infected
mice (E) treated with CI-1040 or DMSO-mock con-
trol. Number of IL-12p40-producing cells (F and
G) as determined by Elispot analysis of total drain-
ing lymph node cells. Western blot of splenic
lysates probed with anti-phosphorylated ERK1/2
and anti-total ERK1/2 (H). Data from at least three
different experiments; error bars denote SEM;
*P 0.05.
1824 Boggiatto et al
AJP May 2009, Vol. 174, No. 5
L. amazonensis amastigote infection of BMDC promotes
increased ERK1/2 activation in the absence of additional
activating stimuli. In contrast to these findings, Xin et al
observed that both L. amazonensis promastigotes and
amastigotes both reduce ERK1/2 phosphorylation in the
presence or absence of activating stimuli.
15
These differ
-
ences could be explained by the time points chosen for
analysis. Based on our data, the 6.5-hour time point
occurs after the observed peak in ERK1/2 phosphoryla-
tion by both L. amazonensis amastigotes (7 minutes) and
promastigotes (4.5 hours).
We report here that inhibition of ERK phosphorylation
with the ERK inhibitor PD98059 before BMDC infection
with L. amazonensis amastigotes leads to an enhanced
CD40 surface expression and IL-12p40 production (Fig-
ure 4, A and B). In vivo, treatment of mice with the orally
available ERK inhibitor CI-1040 enhanced CD40 surface
expression of CD11c
DC (Figure 4, C and D), but did
not result in an increased number of IL-12p40-producing
cells collected from the DLNs of L. amazonensis-infected
mice (Figure 4F). We hypothesize that the observed de-
crease in the number of IL-12p40-producing cells from L.
amazonensis-infected mice, in the presence of ERK inhi-
bition, may be a result of the lower number of CD11c
cells found in the DLN (CD11c
events with no treatment,
2818 vs. CD11
events with CI-1040, 1225). CI-1040-
treatment of Raf-transformed hematopoietic cells leads to
increased sensitivity to apoptosis.
30
We suggest that sys
-
temic ERK inhibition via treatment with this inhibitor may
adversely affect the survival of monocytes migrating into
the site of infection and later into the DLN. A closer
analysis of IL-12p40 production of individual CD11c
events from the DLNs of infected mice may be required to
determine the effects of ERK inhibition on the production
of this cytokine.
Our observation of a more mature DC phenotype fol-
lowing treatment of L. amazonensis-infected C3HeB/FeJ
mice with the ERK inhibitor CI-1040 complements work
from our laboratory showing that ERK inhibition can pro-
mote parasite killing in L. amazonensis-infected macro-
phages in vitro.
19
The data are also consistent with work
indicating that inhibition of ERK slows disease progres-
sion of L. amazonensis-infected BALB/c mice.
18
Although
we did not observe a restoration in IL-12p40 production
during in vivo L. amazonensis infection, ERK inhibition did
enhance CD40 surface expression. Previously, it has
been shown that CD40-CD40L interactions are important
during L. amazonensis infection, as deficiencies in this
interaction lead to increased susceptibility to infection.
10
The work presented here indicates that L. amazonensis
specifically targets CD40 expression in vivo, and that we
can restore CD40 surface expression by inhibiting phos-
phorylation of ERK. We suggest that the mature pheno-
type restored to CD11c
DC via ERK inhibition would
promote the development of a productive CD4
T cell
response during L. amazonensis infection; however, fur-
ther studies are necessary. The work presented here
furthers our understanding of host-parasite amastigote-
specific interactions and provides evidence for the use of
ERK inhibitors as immunomodulators to directly enhance
the host immune response after Leishmania infection.
Acknowledgments
We thank Pfizer Incorporated, Global Research & Devel-
opment, for providing the ERK inhibitor CI-1040, Mr. Kyle
Metz for his technical assistance, Dr. Marian Kohut for her
critical review of this manuscript, Dr. Bryan Bellaire for
technical advice with immunofluorescence procedures
and microscope use, and Dr. Jeffery Beetham for kindly
proving the anti-LPG CA7AE antibody.
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AJP May 2009, Vol. 174, No. 5
    • "In a CD40 dose-dependent manner, SHP-1 modulates CD40-induced phosphorylation of p38 MAPK and ERK1/2 to favor ERK1/2-dependent IL-10 expression and parasite survival (Figure 2). Xin et al. (2008) and Boggiatto et al. (2009) also demonstrated the importance of the CD40/MAPK signaling pathway in L. amazonensis infection. L. amazonensis upregulated ERK1/2 in dendritic cells, increased IL-10 production and prevented the expression of CD40 and IL-12p40 (one of the subunits of IL-12), leading to the limited activation of dendritic cells and a deficient Th1-type response. "
    [Show abstract] [Hide abstract] ABSTRACT: Leishmania spp and Trypanosoma cruzi are the causative agents of leishmaniasis and Chagas' disease, respectively, two neglected tropical diseases that affect about 25 million people worldwide. These parasites belong to the family Trypanosomatidae and are both obligate intracellular parasites that manipulate host signaling pathways to establish the infection, and also subvert the host innate immune system. Mitogen-activated protein kinases (MAPKs) are serine and threonine protein kinases, highly conserved in eukaryotes, and are involved in signal transduction pathways that are related to modulation of physiological and pathophysiological cell responses. This mini-review highlights the current knowledge about the mechanisms that Leishmania spp and T. cruzi have evolved to target host MAPK signaling pathway, highjack immune response, and in this manner, promote parasite maintenance in the host.
    Full-text · Article · Feb 2016
    • "The response is characterized by a poor initial inflammatory response and ineffective T cell-mediated immunity [2,12]. These results correspond to specific effects on antigen presenting cell (APC) function during infection that could, in part, account for the poor adaptive immune response associated with this parasite [13,14]. However, several studies have demonstrated that simply enhancing a Th1 response either through immune modulation or using animals deficient in IL-10 has, at most, only modest effects on the long-term disease outcomes [2,15,16,17]. "
    [Show abstract] [Hide abstract] ABSTRACT: Footpad infection of C3HeB/FeJ mice with Leishmania amazonensis leads to chronic lesions accompanied by large parasite loads. Co-infecting these animals with L. major leads to induction of an effective Th1 immune response that can resolve these lesions. This cross-protection can be recapitulated in vitro by using immune cells from L. major-infected animals to effectively activate L. amazonensis-infected macrophages to kill the parasite. We have shown previously that the B cell population and their IgG2a antibodies are required for effective cross-protection. Here we demonstrate that, in contrast to L. major, killing L. amazonensis parasites is dependent upon FcRγ common-chain and NADPH oxidase-generated superoxide from infected macrophages. Superoxide production coincided with killing of L. amazonensis at five days post-activation, suggesting that opsonization of the parasites was not a likely mechanism of the antibody response. Therefore we tested the hypothesis that non-specific immune complexes could provide a mechanism of FcRγ common-chain/NADPH oxidase dependent parasite killing. Macrophage activation in response to soluble IgG2a immune complexes, IFN-γ and parasite antigen was effective in significantly reducing the percentage of macrophages infected with L. amazonensis. These results define a host protection mechanism effective during Leishmania infection and demonstrate for the first time a novel means by which IgG antibodies can enhance killing of an intracellular pathogen.
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    • "L. amazonensis utilizes the hosts scaffolding complex and because use of this complex provides spatial specificity, L. amazonensis brings ERK1/2 next to the vacuole it inhabits (Fig. 6b). Use of a PV/endosomal-specific p14/MP1 scaffold complex promotes sustained ERK1/2 phosphorylation [53], alteration of the phagocyte oxidative burst [56] and leads to chronic infection [53, 54, 76, 77]. L. amazonensis has been shown to inhabit the perinuclear region within its PV787980. "
    [Show abstract] [Hide abstract] ABSTRACT: Leishmania amazonensis is an intracellular protozoan parasite responsible for chronic cutaneous leishmaniasis (CL). CL is a neglected tropical disease responsible for infecting millions of people worldwide. L. amazonensis promotes alteration of various signaling pathways that are essential for host cell survival. Specifically, through parasite-mediated phosphorylation of extracellular signal regulated kinase (ERK), L. amazonensis inhibits cell-mediated parasite killing and promotes its own survival by co-opting multiple host cell functions. In this review, we highlight Leishmania-host cell signaling alterations focusing on those specific to (1) motor proteins, (2) prevention of NADPH subunit phosphorylation impairing reactive oxygen species production, and (3) localized endosomal signaling to up-regulate ERK phosphorylation. This review will focus upon mechanisms and possible explanations as to how Leishmania spp. evades the various layers of defense employed by the host immune response.
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