MAJOR ARTICLE
Regulation of Chemokines, CCL3 and CCL4,
by Interferon γ and Nitric Oxide Synthase 2 in
Mouse Macrophages and During Salmonella
enterica Serovar Typhimurium Infection
Bhagawat Chandrasekar, Mukta Deobagkar-Lele, Emmanuel S. Victor, and Dipankar Nandi
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
Background. Interferon γ (IFN-γ) increases the expression of multiple genes and responses; however, the
mechanisms by which IFN-γ downmodulates cellular responses is not well understood. In this study, the repression of CCL3 and CCL4 by IFN-γ and nitric oxide synthase 2 (NOS2) in macrophages and upon Salmonella
typhimurium infection of mice was investigated.
Methods. Small molecule regulators and adherent peritoneal exudates cells (A-PECs) from Nos2−/−mice were
used to identify the contribution of signaling molecules during IFN-γ–mediated in vitro regulation of CCL3,
CCL4, and CXCL10. In addition, infection of bone marrow–derived macrophages (BMDMs) and mice (C57BL/6,
Ifn-γ−/, and Nos2−/−) with S. typhimurium were used to gain an understanding of the in vivo regulation of these
chemokines.
Results. IFN-γ repressed CCL3 and CCL4 in a signal transducer and activator of transcription 1 (STAT1)–
NOS2–p38 mitogen activated protein kinase ( p38MAPK)–activating transcription factor 3 (ATF3) dependent
pathway in A-PECs. Also, during intracellular replication of S. typhimurium in BMDMs, IFN-γ and NOS2 repressed CCL3 and CCL4 production. The physiological roles of these observations were revealed during oral infection of mice with S. typhimurium, wherein endogenous IFN-γ and NOS2 enhanced serum amounts of tumor
necrosis factor α and CXCL10 but repressed CCL3 and CCL4.
Conclusions. This study sheds novel mechanistic insight on the regulation of CCL3 and CCL4 in mouse macrophages and during S. typhimurium oral infection.
Keywords. interferon γ; nitric oxide; nitric oxide synthase 2; macrophages; infection; chemokines; cytokine.
Interferon γ (IFN-γ) is produced by activated T cells,
natural killer cells, and macrophages and demonstrates
the following pleiotropic effects: antiviral, antibacterial,
definition of T-helper subsets, induction of major histocompatibility complex molecules, cell survival, and
others [1, 2]. IFN-γ is important for immune cell responses against infectious microbes, and individuals
Received 25 September 2012; accepted 11 December 2012; electronically published 21 February 2013.
Correspondence: Dipankar Nandi, Professor, Department of Biochemistry, FE14
Biological Sciences Building, Indian Institute of Science, Bangalore 560012
([email protected]).
The Journal of Infectious Diseases 2013;207:1556–68
© The Author 2013. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
[email protected].
DOI: 10.1093/infdis/jit067
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with loss of IFN-γ or its receptor show greater susceptibility to less virulent pathogens [3]. The binding
of IFN-γ to its cognate receptors on target cells leads
to activation of the Janus kinase–signal transducer and
activator of transcription ( JAK-STAT) pathway, which
is important in modulating gene expression [4].
Stat1 −/− mice are also highly susceptible to Listeria
monocytogens and viral infections [5]. Therefore, IFNγ and its downstream signaling molecules in the host
are important in the regulation of immune responses,
especially against infectious diseases.
IFN-γ–mediated regulation of cytokines and chemokines is important in modulating inflammatory responses. Upon intraperitoneal infection of mice with
Salmonella enterica serovar Typhimurium (S. typhimurium), endogenous IFN-γ increases serum amounts
of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α)
[6]. Chemokines are secreted by a variety of cells and are important for several inflammatory responses. IFN-γ induces the
expression of chemokine (C-C) ligand 2 (CCL2), CCL5, chemokine (CXC) ligand 9 (CXCL9), and CXCL10 in bone
marrow–derived macrophages [7]. Importantly, in a model of
experimental autoimmune uveitis, dysregulation of a number
of chemokines in the absence of IFN-γ is observed and is correlated with altered cellular infiltration in the eyes [8].
However, the mechanisms by which IFN-γ modulates the expression of chemokines are not well understood, and is the
focus of this study. Two types of mouse macrophages were
used: unstimulated adherent peritoneal exudate cells (A-PECs)
and bone marrow–derived macrophages (BMDMs). An initial
screen identified CCL3 to be lowered upon IFN-γ treatment in
A-PECs (data not shown). Further studies demonstrated that
CCL3 and its paralog, CCL4, which shares approximately 68%
amino acid sequence similarity, were repressed with IFN-γ;
however, CXCL10 was induced. CXCL10 is important in regulating T-cell responses and trafficking [9], whereas CCL3 and
CCL4 attract a variety of leukocytes in vitro [10, 11]. Enhanced
CCL3 amounts are also found in several autoimmune diseases
[12–14], and it has been shown to play protective roles in a
variety of infectious diseases [15–18]. Interestingly, CCL3 and
CCL4, along with CCL5, share a common receptor, chemokine (C-C) receptor 5 (CCR5), which is a coreceptor for
human immunodeficiency virus (HIV) on CD4+ T cells and
macrophages [19, 20]. In addition, CCL3 and CCL5 also bind
to CCR1 and CCR3. Notably, CCL3, CCL4, and CCL5 are secreted more in HIV-exposed individuals and are known to
functionally inhibit the entry of the virus into host CD4+
T cells [19, 21]. Hence, it is important to understand the regulation of CCL3 and CCL4.
In this study, the mechanisms by which IFN-γ represses
CCL3 and CCL4 in A-PECs were investigated and the functional roles of STAT1, nitric oxide synthase 2 (NOS2), p38
mitogen-activated protein kinases ( p38MAPK), and the activating transcription factor 3 (ATF3) were elucidated. These
findings were extended to an infection scenario using oral inoculation of mice with S. typhimurium. Overall, this study
reveals in vitro and in vivo roles of IFN-γ and NOS2 in the
regulation of CCL3 and CCL4.
METHODS
Chemicals and Other Reagents
Recombinant mouse IFN-γ was obtained from PeproTech
Asia. Lipopolysaccharide from Escherichia coli serotype O55:
B5 (LPS), SP600129 (SP), NG-methyl-L-arginine (LNMA),
S-nitroso-N-acetyl-penicillamine (SNAP), fludarabine (F), Larginine (L-Arg), and L-norvaline (Nor) were obtained from
Sigma Aldrich. SB202190 (SB) was obtained from Merck–
Millipore. Taurolidine was obtained from Enzo Life Sciences.
Isolation and Culturing of Resident A-PECs
A-PECs were isolated by injecting 0.32 M sucrose solution into
the mouse peritoneal cavity; the recovered solution was centrifuged at 500 g for 10 minutes [22]. The cells were resuspended
in Roswell Park Memorial Institute medium 1640 (Sigma) supplemented with 5% heat-inactivated fetal calf serum (Invitrogen), 5 μM β-mercaptoethanol (Sigma), 100 μg/mL penicillin,
250 μg/mL streptomycin, and 50 μg/mL gentamicin (Himedia
Laboratories, India) and plated at a cell density of approximately 2 × 105 cells per well in a 96-well plate. The cells were left to
adhere for 45 minutes at 37°C in a humidified carbon dioxide
incubator (Sanyo) and washed 3 times with phosphate-buffered
saline (PBS; Sigma). A similar procedure was used to obtain
and work with adherent PECs from mice treated with 4%
Brewer’s thioglycollate (TG) medium for 4–5 days.
Cytokine and Chemokine ELISAs
The amounts of cytokines (IFN-γ and TNF-α) in the supernatants and sera were estimated using enzyme-linked immunosorbent assay (ELISA) kits from eBioscience. The amounts of
chemokines (CCL3, CCL4, and CXCL10) in the supernatants
and sera were quantified using ELISA kits from PeproTech.
The manufacturer’s protocols were followed for quantification along with appropriate standards (approximately 31–1000
pg/mL). 3,3′,5,5′-Tetramethylbenzidine was used as the substrate, and optical density readings were obtained at 450 nm
using the VersaMax ELISA plate reader.
Bacterial Strain and Culture
S. typhimurium NCTC 12023 were cultured in Luria broth
and streaked on Salmonella-Shigella (SS) agar plates [6]. A
single colony from the plate was cultured overnight in 3 mL
sterile Luria broth (LB) and used to inoculate 50 mL sterile LB
at 0.2% and grown for 3–4 hours (log phase culture) or overnight (stationary phase culture) at 37°C, 160 rpm. Bacterial
culture was centrifuged, washed with PBS, and resuspended in
PBS for further experiments.
Mice, Infections, and Colony Forming Units (CFU) Analysis
C57BL/6, C57BL/6.Ifnγ −/− (Ifnγ −/−), and C57BL/6.Nos2 −/−
(Nos2 −/−) mice (aged approximately 6–8 weeks) were obtained
from the Central Animal Facility, Indian Institute of Science
(IISc). The mice received humane care, and experiments were
performed according to approved procedures issued by the institutional animal ethics committee. Mice were orally fed approximately 108 CFU of S. typhimurium in a final volume of
0.5 mL PBS. On day 4 post infection, mice were sacrificed,
and blood and other organs were collected. The organs
were weighed and homogenized in PBS; CFU were enumerated on SS agar plates. Blood collected by cardiac puncture
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immediately upon sacrificing the mice was allowed to clot and
centrifuged. Serum was separated and stored at −20°C for
further analysis.
Statistical Analysis
All data were plotted using GraphPad Prism 5 software. The
significance was obtained by performing a 2-tailed unpaired
t test. P values <.05, .01, and .001 were represented as *, **,
and ***, respectively. Also, significance was calculated and represented with respect to untreated cells (Figures 1–5) or control
mice (Figures 6 and 7) at the same time point in each figure.
Detailed descriptions of methods, namely nitrite estimation,
cell viability, reverse-transcription polymerase chain reaction
(RT-PCR), estimation of intracellular protein amounts, siRNAmediated knockdown, and histological analysis, are included in
the Supplementary Methods section.
RESULTS
IFN-γ Represses CCL3 and CCL4 in A-PECs
Initial experiments were designed to study the effects of IFN-γ
on A-PECs. The addition of IFN-γ did not affect cell survival
(Figure 1A); however, Cxcl10 was rapidly induced. Interestingly, the transcripts of Ccl3 and Ccl4 were repressed at later time
points (Figure 1B) and further quantification using quantitative RT-PCR is required; however, these changes were validated at the protein level using ELISA. Although CXCL10, a
well-known IFN-γ–induced chemokine [10], was induced
(Figure 1C), CCL3 and CCL4 were repressed in the supernatant of cells treated with different doses of IFN-γ (Figure 1F
and I). Also, CXCL10 was induced in the supernatants of
A-PECs as early as 6 hours post IFN-γ treatment, and levels
were maintained up to 36 hours (Figure 1D and E). On the
other hand, CCL3 and CCL4 amounts were repressed later,
that is, 24 hours post IFN-γ treatment and remained repressed
for 36 hours (Figure 1G, H and J, K). Therefore, in response
to IFN-γ in A-PECs, the kinetics of repression of the transcripts and secreted amounts of CCL3 and CCL4 were delayed
as compared with CXCL10 induction.
NOS2 Mediates Repression of CCL3 and CCL4 by IFN-γ
As some of the responses to IFN-γ are nitric oxide (NO) dependent [24], the possible role of this molecule in regulating
chemokines was evaluated. Nitrite was induced in response to
IFN-γ in the supernatants of A-PECs (Figure 2A). To study
the functional contribution of NO, an inhibitor of NO synthases, LNMA, was used [25]. Studies with this inhibitor
clearly demonstrate that NO in macrophages does not affect
CXCL10 induction but represses CCL3 and CCL4 amounts
(Supplementary Figure 2A–D). Of the 3 isoforms of NOS,
primary macrophages are known to express NOS2 [26]; therefore, the role of NOS2 in the repression of CCL3 and CCL4 in
response to IFN-γ was investigated. IFN-γ–induced nitrite in
the supernatants of A-PECs from C57BL/6 mice was absent in
A-PECs from Nos2−/− mice (Figure 2A), clearly establishing
NOS2 as the major source of NO in these cells. RT-PCR analysis of C57BL/6 and Nos2−/− A-PECs treated with IFN-γ revealed that induction of Irf1 and Cxcl10 transcripts was
similar in both strains of mice (Figure 2B). Although induction of CXCL10 was unaffected (Figure 2C), the repression of
CCL3 and CCL4 by IFN-γ observed in C57BL/6 A-PECs was
abolished in Nos2 −/− A-PECs both at the transcript level
(Figure 2B) and in culture supernatants (Figure 2D and E).
Activation of p38MAPK Regulates IFN-γ–Mediated CCL3 and
CCL4 Repression
MAPKs are known to play crucial roles in regulating IFN-γ responses in macrophages [7]. The roles of the 2 stress-induced
kinases, p38MAPK and c-Jun N-terminal kinase ( JNK), were
investigated. In response to IFN-γ, intracellular amounts of
p-JNK were not significantly altered (data not shown); however,
the amounts of intracellular p-p38MAPK increased significantly
with time post IFN-γ treatment in A-PECs (Figure 3A). To evaluate the functional contribution of p38MAPK, a well-known inhibitor, SB202190 [27], was used. SB202190 did not affect the
induction of CXCL10 (Figure 3D); however, the repression of
CCL3 and CCL4 in the supernatants of A-PECs by IFN-γ was
rescued by SB202190 (Figure 3E and F).
ATF3 Mediates Repression of CCL3 and CCL4 by IFN-γ
STAT1 Activation is Critical for IFN-γ–Mediated Regulation of
CCL3, CCL4, and CXCL10
Next, the role of the canonical JAK-STAT signaling pathway
in response to IFN-γ was evaluated. The intracellular
phospho-STAT1 ( p-STAT1) amounts increased with time in
A-PECs treated with IFN-γ (data not shown). This increase in
intracellular p-STAT1 amounts upon IFN-γ treatment was
abolished in the presence of fludarabine, a STAT1 inhibitor
[23] (Supplementary Figure 1A). Fludarabine treatment inhibited CXCL10 induction as well as repression of CCL3 and
CCL4 in the presence of IFN-γ (Supplementary Figure 1D–F).
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Analysis of the promoters of mouse Ccl3 and Ccl4 revealed the
presence of a consensus-binding sequence to ATF3 (Figure 4A).
Also, addition of IFN-γ led to an increase in the intracellular
amounts of ATF3 in A-PECs with time (Figure 4B). To understand the functional contributions by ATF3, experiments with
an inducer, taurolidine [28], were performed. Addition of taurolidine induced ATF3 amounts in A-PECs (Supplementary
Figure 2E) and repressed CCL3 and CCL4, but not CXCL10, in
C57BL/6 (Supplementary Figure 2F, G, and H) and Nos2−/−
(Supplementary Figure 2I, J, and K) A-PECs. To confirm the
role of ATF3 in IFN-γ–mediated repression of CCL3 and CCL4,
Figure 1. Interferon γ (IFN-γ) induces CXCL10 but represses CCL3 and CCL4 in adherent peritoneal exudates cells (A-PECs) in a dose- and timedependent manner. A, Changes in the viability of A-PECs from C57BL/6 mice upon treatment with different doses of IFN-γ for 24 hours. B, Kinetic
analysis of Cxcl10, Ccl3, Ccl4, and Hprt using reverse-transcription polymerase chain reaction. The amounts of CXCL10 (C ), CCL3 (F ), and CCL4 (I ) in
the supernatants of A-PECs treated with different doses of IFN-γ for 24 hours. The amounts of CXCL10 (D and E ), CCL3 (G and H ), and CCL4 (J and K )
present in the supernatants of A-PECs treated without or with IFN-γ (5 U/mL), respectively, at indicated time points. Data are represented as mean ±
standard error from 2 independent experiments. Significance is represented with respect to untreated controls.
siRNA-mediated knockdown of ATF3 in A-PECs was performed (Figure 4E). Knockdown of ATF3 did not affect
IFN-γ–induced nitrite and CXCL10 amounts (Figure 4F and
G); however, reduced repression of CCL3 and CCL4 by IFN-γ
was observed upon ATF3 knockdown (Figure 4H and I).
IFN-γ and NOS2 Repress CCL3 and CCL4 During
S. typhimurium Infection in BMDMs
Having established the mechanism of regulation of CCL3 and
CCL4 in A-PECs (Figure 2F), the roles of IFN-γ and NOS2
were studied during intracellular replication of S. typhimurium
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Figure 2. Interferon γ (IFN-γ) mediated repression of CCL3 and CCL4 is nitric oxide synthase 2 dependent. A, Nitrite amounts in the supernatants of
adherent peritoneal exudates cells (A-PECs) from C57BL/6 and Nos2−/− mice after 24 hours of treatment with or without 5 U/mL of IFN-γ. B, Reversetranscription polymerase chain reaction analysis of expression of indicated genes post 24 hours of treatment with or without IFN-γ (5 U/mL) in A-PECs
from C57BL/6 and Nos2−/− mice. The amounts of CXCL10 (C), CCL3 (D), and CCL4 (E) present in the supernatants of A-PECs from C57BL/6 and
Nos2−/− mice after 24 hours of treatment with or without 5 U/mL of IFN-γ. Data are represented as mean ± standard error from 3 independent
experiments. Significance is represented with respect to untreated controls. F, A schematic model depicting the pathway leading to regulation of
chemokines CXCL10, CCL3 and CCL4 by IFN-γ in A-PECs is shown.
in BMDMs. Addition of IFN-γ inhibited intracellular replication
of S. typhimurium (Figure 5A) and repressed the S. typhimurium
infection–induced production of CCL3 and CCL4 (Figure 5C
and D), but not CXCL10 (Figure 5B). Also, higher CFU were
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recovered upon S. typhimurium infection of BMDMs from
Nos2−/− mice (Figure 5E). Notably, the absence of NOS2 led to
increased amounts of CCL3 and CCL4, but not CXCL10, in the
supernatants upon S. typhimurium infection (Figure 5F–H).
Figure 3. Interferon γ (IFN-γ) induced p-p38 mitogen-activated protein kinases ( p-p38MAPK) functionally represses CCL3 and CCL4. Kinetic analysis
of the fold change in the intracellular amounts of p-p38MAPK in adherent peritoneal exudates cells (A-PECs from C57BL/6 (A) and Nos2−/− (B) mice
treated with 5 U/mL IFN-γ. The amounts of nitrite (C), CXCL10 (D), CCL3 (E), and CCL4 (F) present in the supernatants of A-PECs of C57BL/6 treated
with 5 U/mL IFN-γ in the presence or absence of p38MAPK inhibitor SB202190 (SB for 24 hours. Data are represented as mean ± standard error from 2
independent experiments. Significance is represented in comparison to untreated control in each panel.
IFN-γ and NOS2 Repress CCL3 and CCL4 In Vivo upon
S. typhimurium Infection of Mice
Next, the regulation of CXCL10, CCL3, and CCL4 during oral
infection of mice with S. typhimurium was investigated. Mice
lacking IFN-γ or NOS2 had higher CFUs in different organs
on day 4 post S. typhimurium oral infection compared with
C57BL/6 (Figure 6A–D). Also, histopathological examination
of livers revealed greater damage in Ifn-γ−/− and Nos2−/− mice
upon infection when compared with C57BL/6-infected mice
(Figure 6E). Consistent with previous studies [6, 29], both
Ifn-γ−/− and Nos2−/− mice were more susceptible to S. typhimurium infection compared to C57BL/6 mice.
Importantly, analysis of uninfected and infected mice sera
revealed that induction of IFN-γ in sera upon infection was
similar between C57BL/6 and Nos2−/− mice (Figure 7A).
However, Ifnγ−/− and Nos2−/− mice had significantly less
TNF-α and CXCL10 induction compared with C57BL/6 mice
upon infection (Figure 7B and C). Most importantly, amounts
of CCL3 and CCL4 were significantly larger in both Ifnγ−/−
and Nos2−/− mice sera upon infection compared to C57BL/6
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Figure 4. ATF3 functionally represses CCL3 and CCL4 in response to interferon γ (IFN-γ). A, Bioinformatic analysis of the consensus binding sequence for ATF3 in the promoters of mouse Ccl3 and Ccl4. Kinetic analysis of ATF3 amounts in response to 5 U/mL of IFN-γ in adherent peritoneal
exudates cells (A-PECs) from C57BL/6 (B) and Nos2−/− (C) mice. Regulation of ATF3 in A-PECs from C57BL/6 in the absence or presence of inhibitors
fludarabine (F ), NG-methyl-L-arginine (LNMA), and SB202190 (SB) after 24 hours upon IFN-γ treatment (D). Amount of intracellular ATF3 (E ) and nitrite
(F ), CXCL10 (G ), CCL3 (H), and CCL4 (I) in the supernatants of A-PECs transfected with scrambled or Atf3 siRNA in the presence or absence of IFN-γ
for 24 hours. Data are represented as mean ± standard error from 2 independent experiments. Significance is represented in comparison with untreated
control in each panel.
mice (Figure 7D and E). Therefore, endogenous IFN-γ and
NOS2 increase TNF-α and CXCL10 but repress CCL3 and
CCL4 amounts in vivo during S. typhimurium infection of
mice.
DISCUSSION
In this study, the critical roles of IFN-γ and NOS2 in repressing the production of chemokines CCL3 and CCL4 are
shown, both in vitro and in vivo. Importantly, the repression
of CCL3 and CCL4 was specific to IFN-γ, but not LPS (data
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not shown). Also, repression of CCL3 and CCL4 amounts affected by IFN-γ was observed in primary unstimulated macrophages such as A-PECs (Figure 1) and BMDMs (Figure 5),
but not activated macrophages, for example, TG-elicited PECs
(data not shown). Thus, the repression of CCL3 and CCL4 is
ligand and cell specific. Also, LPS is known to stimulate CCL3
production by elicited macrophages [30], which are distinct
from unstimulated A-PECs [31]. Studies involving unstimulated A-PECs may be useful in gaining an understanding of
the macrophage responses that occur during early stages of
immune challenge. This aspect of cell specificity is important
because the initial phase of the immune response, which
Figure 5. Interferon γ (IFN-γ) and nitric oxide synthase 2 repress Salmonella typhimurium infection–induced CCL3 and CCL4 in bone marrow–derived
macrophages (BMDMs). The amount of intracellular colony-forming unit (CFU) burden (A) and CXCL10 (B), CCL3 (C), and CCL4 (D) in the supernatants
after 18 hours of S. typhimurium infection of BMDMs from C57BL/6 mice treated without or with 100 U/mL IFN-γ. The amount of intracellular CFU
burden (E) and CXCL10 (F), CCL3 (G), and CCL4 (H) in the supernatants after 18 hours of S. typhimurium infection of BMDMs from C57BL/6 and Nos2−/−
mice. Data are represented as mean ± standard error from 3 independent experiments. Significance is represented with respect to C57BL/6 controls.
involves unstimulated macrophages, may be geared towards
generating enhanced T-cell responses, as shown with higher
levels of CXCL10 and lower levels of CCL3 and CCL4.
However, subsequent responses by activated macrophages may
be more inflammatory due to a lack of repression of CCL3
and CCL4.
IFN-γ is known to repress CCL3 and CCL4 in the macrophage cell line ANA-1 [32]; however, the underlying molecular
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Figure 6. Ifnγ−/− and Nos2 −/− mice show increased colony-forming unit (CFU) and cellular infiltration to oral infection with Salmonella typhimurium.
CFU burden on day 4 post oral dosing with approximately 108 S. typhimurium in Peyer’s patches (A), mesenteric lymph node (B), spleen (C), and liver (D)
from C57BL/6, Ifnγ−/−, and Nos2−/− mice. Data are represented as mean ± standard error from 4 independent experiments. Significance is represented
with respect to C57BL/6 mice. Histological examination of liver sections from uninfected and S. typhimurium–infected livers of C57BL/6, Ifnγ−/−, and
Nos2 −/− mice (E). Sections were stained with hematoxylin and eosin, and images were obtained at 200 × in a light microscope fitted with Nikon
Coolpix camera. Images are representative of 5 or more sections per group.
mechanisms are not understood. Although CXCL10 is known
to be induced in a STAT1-dependent manner in macrophages [33], STAT1-dependent repression of CCL3 and CCL4
in response to IFN-γ is shown. Also, IFN-γ–induced NO is
known to modulate a variety of responses [24], and there are
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conflicting reports on the regulation of CCL3 by NO in different cells [34, 35]. Importantly, the repression of CCL3 and
CCL4 in response to IFN-γ treatment was not observed upon
inhibition of NOS activity by LNMA (Supplementary
Figure 2A–D) and in cells lacking NOS2 (Figure 2). A
Figure 7. Interferon γ (IFN-γ) and nitric oxide synthase 2 repress the production of CCL3 and CCL4 in sera upon Salmonella typhimurium infection in
mice. The amounts of IFN-γ (A), tumor necrosis factor-α (B), CXCL10 (C), CCL3 (D), and CCL4 (E) present in the sera from uninfected and 4 days post S.
typhimurium–infected C57BL/6, Ifn-γ−/−, and Nos2−/− mice. Data are represented as mean ± standard error from 4 independent experiments. Significance is represented with respect to untreated C57BL/6 mice.
possibility existed that the inhibition of NOS2 led to arginine
accumulation or effects of its catabolism due to arginase.
However, addition of excess arginine or inhibition of arginase
using the inhibitor norvaline increased amounts of IFN-γ–
induced nitrite [36] but did not affect the lowering of CCL3 or
CCL4 (Supplementary Figure 1G–M). Thus, NO production
by NOS2 in response to IFN-γ plays a functional role in the
repression of CCL3 and CCL4. It is important to note that NO
alone, as provided by the NO donor SNAP, did not affect the
basal production of CCL3, CCL4, and CXCL10 (data not
shown). These results demonstrate that a combination of IFNγ–specific signals, including the production of NO, leads to repression of CCL3 and CCL4.
IFN-γ–induced MAPKs are known to regulate chemokines
as well as other IFN-γ–inducible genes in BMDMs [7]. Also,
p38MAPK is known to stabilize CCL3 in response to LPS in
human peripheral blood mononuclear cells [27]. Therefore,
the possible role of MAPKs in regulating CCL3, CCL4, and
CXCL10 in A-PECs was studied. Inhibitor studies revealed
that activated p38MAPK, but not JNK, was important in
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IFN-γ–mediated repression of CCL3 and CCL4, but not
CXCL10 induction (Figure 3 and data not shown). The transcription factor ATF3 is known to be induced in response to
IFN-γ in keratinocytes [37]. Also, ATF3 is known to be a transcriptional repressor of CCL4 in mouse macrophages [38] and
it is inducible by NO and p38MAPK [39, 40]. The functional
roles of ATF3 were addressed using two approaches: addition
of taurolidine, a known ATF3 inducer that exhibits anti-inflammatory and antitumorigenic activities [28] (Supplementary Figure 2E–K) and lowering of ATF3 using specific siRNAs
(Figure 4). These data are consistent with a role of IFN-γ–
induced ATF3 acts as a transcriptional repressor of CCL3 and
CCL4.
Since STAT1 inhibition reduced IFN-γ–mediated NO induction (Supplementary Figure 1B), NOS2 was downstream to
STAT1. Inhibition of p38MAPK did not affect IFN-γ–induced
NO (Figure 3C); however, activation and sustenance of
p38MAPK was reduced in Nos2−/− A-PECs compared with
C57BL/6 upon IFN-γ treatment (Figure 3A and B). There are
conflicting reports on the regulation of NOS2 by p38MAPK
in macrophages [7,41]; however, the observation that IFN-γ–
regulated NOS2 increases and sustains p38MAPK activation is
novel. Also, induction of ATF3 is dependent on STAT1, NO,
p38MAPK, and NOS2 (Figure 4B–D). Thus, induction of
ATF3 is downstream and represses CCL3 and CCL4. Overall,
our study highlights the fact that IFN-γ and its signaling components, including NOS2, are important in the regulation of
CCL3 and CCL4 (Figure 2F).
Next, we extended our study from A-PECs to an infection
model using the intracellular pathogen, S. typhimurium. This
aspect is an important area of study as chemokines and their
receptors modulate infection and inflammatory processes.
Chemokine amounts are known to be regulated upon infection of macrophages with Borrelia burgdorferi, Mycobacterium
tuberculosis, and others [42, 43]. CCL3 is important in resisting infections with virulent Cryptococcus neoformans [15] and
Klebsiella pneumoniae [16]. Interestingly, leukocytic infiltration and cytokine amounts are not affected during infection
with K. pneumoniae; however, the phagocytic activity of
Ccl3 −/− macrophages is lower [16]. Also, inheritance of a truncated allele of Ccr5 abrogates cell surface expression and
confers protection against HIV infection [19]. Studies with
mice lacking Ccr5 have shown its role in modulating cytokine
production, lowering T-cell responses, and enhancing resistance to Listeria [44]. CCR5 also lowers the infiltration of lymphocytes and dendritic cells in lungs and draining lymph
nodes upon infection with M. tuberculosis [45]. Also, IFN-γ
and NOS2 are known to be extremely important for defense
against a plethora of intracellular pathogens [26, 46–48];
however, their role in modulation of chemokines is unclear.
HIV infection induces NO, and inhibition of this process
leads to lower CCL3 amounts [35], whereas Trypanosoma
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cruzi–infected cardiac myocytes from Nos2−/− mice produce
higher CCL2, CCL4, and CCL5 amounts [49].
Addition of IFN-γ decreased the CFU and amounts of
CCL3 and CCL4 upon S. typhimurium infection of C57BL/6
BMDMs (Figure 5A–D). Also, infection of Nos2−/−BMDMs
with S. typhimurium resulted in higher CFU and CCL3 and
CCL4 amounts (Figure 5E–H). These observations prompted
us to investigate the contributions of IFN-γ and NOS2 in the
regulation of CCL3 and CCL4 in vivo upon S. typhimurium
infection in mice. Mice lacking IFN-γ and NOS2 displayed
more CFU (Figure 6), which is consistent with previous
studies [6, 29]. This study reveals differential roles of IFN-γ
and NOS2 in modulating cytokines and chemokines during in
vivo S. typhimurium infection: increasing TNF-α and CXCL10
but lowering CCL3 and CCL4. The observation that Ifnγ−/−
and Nos2−/− mice infected with S. typhimurium had significantly higher amounts of CCL3 and CCL4 in sera compared
with C57BL/6 mice (Figure 7D and E) is in agreement with
the greater infiltration of leukocytes in tissues, for example,
liver, in these mice (Figure 6). Although both IFN-γ and
NOS2 are known to restrict S. typhimurium infection, the
functional role of CCL3 and CCL4 during S. typhimurium infection needs to be studied. It is possible that the absence of
IFN-γ or NOS2 leads to greater recruitment of leukocytes, including CCR5+ cells, as a result of higher amounts of CCL3
and CCL4. It is known that blockade of CCR1 and CCR5
using an antagonist, N-terminal methionylated RANTES,
lowers cellular infiltration and bone loss during periodontal
infections [50]. Therefore, it is possible that greater leukocytic
infiltration into tissues may lead to more host cell damage, as
observed in the livers of S. typhimurium–infected Ifnγ−/− and
Nos2−/− mice (Figure 6). Thus, tight regulation of CCL3 and
CCL4 by IFN-γ and NOS2 may be important for the host response during infections with an intracellular pathogen such
as S. typhimurium.
Overall, our study with unstimulated A-PECs, BMDMs,
and in vivo infection of mice with S. typhimurium identifies
IFN-γ and NOS2 as key regulators of the chemokines CCL3
and CCL4. A better understanding of the regulation of these
molecules may provide insights into modulation of the host
immune response to effectively ameliorate several diseases in
which CCL3 and CCL4 contribute significantly, including autoimmune disorders such as diabetes and infections caused by
C. neoformans, K. pneumoniae, HIV, and others.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases
online (http://jid.oxfordjournals.org/). Supplementary materials consist of
data provided by the author that are published to benefit the reader. The
posted materials are not copyedited. The contents of all supplementary
data are the sole responsibility of the authors. Questions or messages
regarding errors should be addressed to the author.
Notes
Acknowledgements. The Central Animal Facility, IISc, is acknowledged for providing mice. We appreciate the help of Prof A.G. Samuelson
for use of the fluorescence plate reader facility, which is funded by the
Department of Science and Technology. All members of our laboratory
are acknowledged for their support and encouragement, especially Suni
Chacko for performing the initial array that led to this study.
Financial support. This work was supported by the grant BT/
PR7240/INF/22/52/2006 from the Department of Biotechnology (DBT),
India. BC is thankful to DBT for student fellowship and MDL received
SPM fellowship from Council of Scientific and Industrial Research (CSIR),
India.
Potential conflict of interest. The authors declare no conflict of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed
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