Cx3cr1 deficiency in mice attenuates hepatic granuloma formation during acute schistosomiasis by enhancing the M2-type polarization of macrophages
Published by The Company of Biologists Ltd | Disease Models & Mechanisms
Cx3cr1 deficiency in mice attenuates hepatic granuloma formation during acute schistosomiasis by enhancing the M2-type polarization of macrophages
Lin Ran 1 2
Qilin Yu 1
Shu Zhang 1
Fei Xiong 1
Jia Cheng 1
Ping Yang 1
Jun-Fa Xu 0
Hao Nie 6
Qin Zhong 2
Xueli Yang 2
Fei Yang 6
Quan Gong 5
Michal Kuczma 4
Piotr Kraj 4
Weikuan Gu 3
Bo-Xu Ren ( 2
Cong-Yi Wang ) 0 1 2
0 Department of Clinical Immunology, Institute of Laboratory Medicine, Guangdong Medical College , No. 1 Xincheng Road, Dongguan 523808 , China
1 The Center for Biomedical Research, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology , 1095 Jiefang Avenue, Wuhan 430030 , China
2 Department of Molecular Biology, Medical College of Yangtze University , 1 Nanhuan Road, Jingzhou, Hubei 434023 , China
3 Department of Orthopedic Surgery and BME, Campbell-Clinic, University of Tennessee, Health Science Center , Memphis, TN 38163 , USA
4 The Center for Biotechnology and Genomic Medicine, Georgia Regents University , 1120 15th Street, Augusta, GA 30912 , USA
5 Department of Immunology, Medical College of Yangtze University , 1 Nanhuan Road, Jingzhou, Hubei 434023 , China
6 Clinical and Molecular Immunology Research Center, Medical College of Yangtze University , 1 Nanhuan Road, Jingzhou, Hubei 434023, China,
Acute schistosomiasis is characterized by pro-inflammatory responses against tissue- or organ-trapped parasite eggs along with granuloma formation. Here, we describe studies in Cx3cr1−/− mice and demonstrate the role of Cx3cr1 in the pathoetiology of granuloma formation during acute schistosomiasis. Mice deficient in Cx3cr1 were protected from granuloma formation and hepatic injury induced by Schistosoma japonicum eggs, as manifested by reduced body weight loss and attenuated hepatomegaly along with preserved liver function. Notably, S. japonicum infection induced high levels of hepatic Cx3cr1 expression, which was predominantly expressed by infiltrating macrophages. Loss of Cx3cr1 rendered macrophages preferentially towards M2 polarization, which then led to a characteristic switch of the host immune defense from a conventional Th1 to a typical Th2 response during acute schistosomiasis. This immune switch caused by Cx3cr1 deficiency was probably associated with enhanced STAT6/PPAR-γ signaling and increased expression of indoleamine 2,3-dioxygenase (IDO), an enzyme that promotes M2 polarization of macrophages. Taken together, our data provide evidence suggesting that CX3CR1 could be a viable therapeutic target for treatment of acute schistosomiasis.
CX3CR1; Schistosomiasis; Granuloma formation; Macrophage; STAT-6; PPAR-γ
A characteristic pathological manifestation of schistosomiasis is
the granulomatous response against tissue- or organ-trapped
parasite eggs (ova) (Pearce and MacDonald, 2002). In particular,
the formation of hepatic egg granulomas and secondary hepatic
fibrosis are the primary cause of death in schistosomiasis
(Vennervald and Dunne, 2004). Soluble egg antigen (SEA)
originating from the eggs of Schistosoma japonicum is potent
enough to evoke pro-inflammatory responses by recruiting
macrophages into the liver, which then initiate granuloma
formation to limit the immune responses against SEA to the
location of the trapped egg in the liver (Burke et al., 2010; Qiu et al.,
2001; Shimaoka et al., 2007). Given that macrophages serve as a
bridge to link innate immunity to adaptive immune responses, they
have now been recognized to play a crucial role in the pathogenesis
of granuloma formation during the course of schistosomiasis
(Behrens, 2008; Christophi et al., 2009; Gordon and Martinez,
2010; Noel et al., 2004; Ragheb and Boros, 1989).
In general, praziquantel is thus far the best therapeutic choice
for treatment of schistosomiasis, although recent studies have
consistently raised concerns about the development of parasite
praziquantel resistance. Furthermore, schistosomes possess the
capability to evade the immune system of the host, which allows
them to survive intravascularly for many years in the face of an
ongoing antiparasite immune response by the infected host (Pearce
and MacDonald, 2002). As a result, sustained aggravation of hepatic
granulomatous inflammatory responses and subsequent fibrosis are
commonly noted in certain patients, even when efficacious
antiparasitic drugs are administered (Cioli and Pica-Mattoccia,
2003). Therefore, a better understanding of the pathoetiologies
underlying granuloma formation during the course of schistosome
infection is essential to develop novel effective therapeutic
strategies for prevention and treatment of hepatic fibrosis.
Previous studies have suggested that chemokines and their
receptors not only coordinate inflammatory infiltration but also
modulate the function of resident immune cells in the setting of
tissue and/or organ injury or infection. In particular, CX3CR1 has
been implicated in the pathogenesis of rheumatoid arthritis,
glomerulonephritis, atopic dermatitis, psoriasis, Crohn’s disease
and atherosclerosis (Ishida et al., 2008). More recently, several
studies have provided evidence supporting the idea that the
expression of Cx3cr1 on monocytes or macrophages promotes
wound healing and fibrotic processes (Martins-Green et al., 2013).
Based on these observations, we thus hypothesized that CX3CR1
signaling in infiltrating macrophages could play a crucial role in the
formation of hepatic egg granulomas after schistosome infection.
To test this hypothesis, B6 mice deficient in Cx3cr1 were infected
with cercariae of S. japonicum. Our data demonstrate that loss of
Cx3cr1 signaling significantly protected mice from hepatic
granuloma formation along with preserved liver function.
Schistosomiasis is a parasitic disease that affects more than 210 million
people worldwide. Its major pathology is the induction of a
proinflammatory response against parasite eggs trapped in tissues or
organs, which leads to the formation of granulomas (nodules of immune
system cells that wall off and contain foreign bodies). In general,
praziquantel is the best therapeutic choice for treatment of infections with
all major schistosome species. However, praziquantel is only effective
against adult worms and requires the presence of a mature antibody
response to the parasite. Furthermore, schistosomes possess the
capability to evade the immune system of the host. As a result,
sustained aggravation of hepatic granulomatous inflammatory
responses and subsequent fibrosis are commonly noted in some
individuals affected by schistosomiasis even when efficacious
antiparasitic drugs are administered.
Recently, it has been reported that the expression of the chemokine
Cx3cr1 on monocytes and macrophages promotes wound healing and
fibrotic processes. In this study, therefore, the authors test the hypothesis
that CX3CR1 signaling in infiltrating macrophages plays a crucial role in
the formation of hepatic granulomas after schistosome infection using
mice infected with Schistosoma japonicum, a well-established model of
human schistosomiasis. The authors report that mice deficient in Cx3xr1
are protected from granuloma formation and hepatic injury induced by
S. japonicum eggs, as manifested by a reduced loss of body weight,
attenuated hepatomegaly and preservation of liver function. Notably,
S. japonicum infection induced high levels of Cx3cr1 expression in the
liver, predominantly by infiltrating macrophages. Moreover, loss of
Cx3cr1 directed macrophages preferentially towards M2 polarization.
This, in turn, led to a characteristic switch of host immune defense in the
setting of acute schistosomiasis from a conventional Th1 to a typical Th2
response. Finally, the authors show that this immune switch was
associated with enhanced STAT6/PPAR-γ signaling and increased
expression of IDO, a tryptophan-metabolizing enzyme that promotes M2
polarization of macrophages.
Implications and future directions
These findings support the hypothesis that Cx3cr1 signaling in infiltrating
macrophages is linked to the formation of hepatic egg granulomas after
schistosome infection in mice. These findings therefore suggest that it
might be possible to attenuate hepatic granuloma formation in people
infected with schistosomes by suppressing the expression of CX3CR1.
Thus, CX3CR1 could be a viable therapeutic target for the treatment of
individuals with acute schistosomiasis.
Loss of Cx3cr1 protects mice against hepatic granuloma
Wild-type (WT) mice and Cx3cr1−/− mice were percutaneously
infected with 30 cercariae of S. japonicum, and the mice were
sacrificed 8 weeks after infection. We first noted that S. japonicum
infection resulted in a significant reduction in body weight in WT
mice, and in sharp contrast, Cx3cr1−/− mice generally looked
healthy as manifested by no statistical change in terms of body
weight (Fig. 1A). Furthermore, WT mice displayed a marked
increase in liver weight as manifested by hepatomegaly (Fig. 1B),
and a similar increase in the colonic weight was also noted in WT
mice after S. japonicum infection as compared with that of Cx3cr1−/−
mice (Fig. 1C).
Next, we sought to examine the impact of S. japonicum infection
on liver function by assaying the activities of alanine transaminase
(ALT) and aspartate transaminase (AST). Both WT and Cx3cr1−/−
mice manifested an increase in ALT and AST activity 8 weeks after
cercariae infection. WT mice showed significantly higher levels of
ALT (Fig. 1D) and AST (Fig. 1E) as compared with that in Cx3cr1−/−
mice, demonstrating that Cx3cr1 deficiency protected mice from
the hepatic injury caused by S. japonicum infection. H&E staining
of liver sections was then carried out to assess granuloma formation.
In accordance with the above results, a significantly smaller
average hepatic granuloma size was observed in Cx3cr1−/− mice
as compared with that of WT control mice (122,118±2376 μm2
versus 35,155±697 μm2, mean±s.e.m., P<0.001, Fig. 2A), and
similar results were also noted in the colon (Fig. 2B). Collectively,
our data support the idea that loss of Cx3cr1 provides protection for
mice against hepatic granuloma formation and functional
impairment induced by S. japonicum eggs.
S. japonicum infection induces high levels of hepatic Cx3cr1
To further demonstrate the role of Cx3cr1 in hepatic granuloma
formation induced by S. japonicum eggs, we then examined hepatic
Cx3cr1 expression in WT mice 8 weeks after cercariae infection;
age matched WT mice in the absence of S. japonicum infection
served as controls. Interestingly, only low levels of Cx3cr1
expression were detected in the liver under physiological
conditions. However, S. japonicum infection induced almost a
7-fold increase in hepatic Cx3cr1 expression (Fig. 3A). To confirm
these data, we conducted immunostaining of liver sections for
Cx3cr1 along with F4/80, a specific marker for macrophages. As
shown in Fig. 3B, Cx3cr1 was almost undetectable in the sections
originating from normal mice, and only some sporadic
F4/80positive macrophages were observed. In sharp contrast, a high
intensity of immunofluorescence specific for Cx3cr1 was noted
within the granuloma in the sections derived from S.
japonicuminfected mice, and a significant accumulation of F4/80-positive
macrophages within the granulomatous area was noted. In
particular, Cx3cr1 immunofluorescence colocalized with F4/80
staining, suggesting that Cx3cr1 was expressed predominantly by
the infiltrating macrophages. Of note, although macrophages were
the major type of infiltrating immune cells, other inflammatory cells
such as neutrophils, lymphocytes and eosinophils were also
detected in the granulomas (data not shown).
Cx3cr1−/− mice manifest a Th2 response against
livertrapped S. japonicum eggs
To dissect the mechanisms by which Cx3cr1 deficiency attenuates
hepatic granuloma formation after S. japonicum infection, we first
examined arginase-1 (Arg-1) and inducible NO synthase (iNOS)
expression in the liver, because enhanced Arg-1 expression is
associated with a Th2 immunity to liver-trapped S. japonicum eggs,
whereas a Th1-mediated response is a feature of increased iNOS
expression (Zhang et al., 2001). Interestingly, no significant
difference in terms of hepatic Arg-1 expression between S.
japonicum-infected mice and control mice was observed
(Fig. 4A). However, more than a 2-fold higher iNOS expression
was noted in S. japonicum-infected WT mice as compared with that
of uninfected WT control mice (Fig. 4B), confirming that hepatic
granuloma formation in acute schistosomiasis is predominantly
mediated by a Th1-based immune response rather than a Th2-related
immunity in WT mice.
Next, we compared the expression of Arg-1 and iNOS between
WT and Cx3cr1−/− mice after S. japonicum infection. Remarkably,
significantly higher levels of Arg-1 were noted in Cx3cr1−/− mice
than in WT mice (Fig. 4C). By contrast, Cx3cr1−/− mice manifested
a 4.8-fold reduction in iNOS expression as compared to WT mice
Fig. 1. Pathological manifestations 8 weeks after S. japonica infection. (A) Body weight changes after S. japonica infection. (B) Liver weight changes.
(C) Colonic swelling and damage after S. japonica infection. (D) ALT levels after S. japonica infection. (E) Results for AST levels. Cx3cr1−/− mice were significantly
protected from hepatic injury and functional impairment mediated by S. japonicum infection as manifested by the reduced body weight loss, attenuated
hepatomegaly along with reserved liver function. A total of 15 mice were analyzed in each study group. *P<0.05; **P<0.01.
(Fig. 4D). Of note, no significant difference in terms of Arg-1
(Fig. 4E) and iNOS expression (Fig. 4F) between WT and Cx3cr1−/−
mice before S. japonicum infection was detected. Taken together,
these data suggest that Cx3cr1 deficiency promotes a characteristic
switch of host immunity against S. japonicum eggs from a typical
Th1 response to a Th2-based reaction during the stage of acute
Lack of Cx3cr1 preferentially induces macrophages toward
To confirm the above data obtained in animal studies, peritoneal
macrophages were isolated from WT and Cx3cr1−/− mice, and then
subjected to stimulation with SEA. SEA induced expression of high
levels of Cx3cr1 in WT macrophages (Fig. 5A). In particular, SEA
displayed a high potency to induce iNOS expression (Fig. 5B),
whereas the expression of Arg-1 was repressed by SEA in
WT macrophages (Fig. 5C). We then compared the expression of
iNOS and Arg-1 between WT and Cx3cr1−/− macrophages. No
perceptible difference in iNOS expression between WT and
Cx3cr1−/− macrophages before SEA stimulation was noted (data
not shown), whereas Cx3cr1−/− macrophages manifested a slightly
higher Arg-1 expression as compared to WT macrophages
(Fig. 5D). However, WT macrophages displayed a 1.5-fold higher
iNOS expression than that of Cx3cr1−/− macrophages 96 h after
SEA stimulation (Fig. 5E), and, by contrast, a 1-fold higher Arg-1
expression was detected in Cx3cr1−/− macrophages as compared
with that of WT macrophages (Fig. 5F). Collectively, these data
support that Cx3cr1−/− macrophages preferentially polarize to a M2
phenotype upon SEA induction.
We next conducted flow cytometry for analysis of CD206
expression, another phenotypic marker for M2 macrophages
(Martinez, 2011). For this purpose, SEA-stimulated macrophages
were first gated for CD11b and F4/80, and CD11b+ F4/80+ cells
were then subjected to analysis of CD206 expression. Indeed, there
was an ∼1.0-fold higher number of Cx3cr1−/− macrophages that
were CD206-positive as compared with the number of WT
macrophages at 96 h after SEA stimulation (Fig. 6A). Moreover,
SEA induced a distinctive morphological change in the
macrophages (Lee et al., 2013). The most striking morphological
differences were noted 96 h after SEA stimulation, in which WT
macrophages displayed M1 morphological features, characterized
by the hemispherical shape along with larger surface area, full
volume and high tension, whereas Cx3cr1−/− macrophages
demonstrated M2 characteristics of stellate shape featured by long
tentacles and a small surface area as well as low tension (Fig. 6B).
Of note, no perceptible morphological difference was noted
Fig. 2. S. japonica egg-induced
granuloma formation. (A) Analysis of
granuloma size in the liver. (B) Analysis
of colonic granuloma size. The number
of granulomas and their size in the
sections were analyzed in both WT and
Cx3cr1−/− mice before and 8 weeks
after S. japonica infection. The graphs
show results after S. japonica infection.
Eight mice were analyzed in each study
group. *P<0.05; **P<0.01.
between WT and Cx3cr1−/− macrophages before SEA stimulation
(data not shown). These results prompted us to examine the
differences in their cytokine secretion. To do this, we assayed
TNFα, IFN-γ, IL-4 and IL-10 production in the culture supernatants at
various time points. In agreement with the above data, Cx3cr1−/−
macrophages secreted significantly higher levels of IL-4 and IL-10
than WT macrophages (Fig. 6C), whereas WT macrophages
secreted significantly higher levels of TNF-α and IFN-γ as
compared to Cx3cr1−/− macrophages (Fig. 6D).
Cx3cr1 deficiency promotes STAT6–PPAR-γ signaling and
We next sought to address how loss of Cx3cr1 skews macrophages
towards M2 polarization upon stimulation with SEA. Given that
STAT6 has been demonstrated to act as an IL-4 signal mediator (Sajic
et al., 2013), we thus first examined the impact of Cx3cr1 deficiency
on the STAT6-PPAR-γ axis, an essential signaling pathway relevant
to the induction of M2 macrophages (Sica and Mantovani, 2012). As
expected, SEA induced more than a 1.5-fold increase in total STAT6
in Cx3cr1−/− macrophages at 96 h after stimulation with SEA as
compared to in control macrophages (Fig. 7A). In line with this
observation, Cx3cr1−/− macrophages manifested significantly higher
levels of phosphorylated (activated) STAT6 ( p-STAT6) than control
macrophages (Fig. 7B). Analysis of the downstream factor PPAR-γ
also revealed that SEA-stimulated Cx3cr1−/− macrophages express
much higher levels of PPAR-γ (Fig. 7C). To further demonstrate that
the enhanced STAT6 and PPAR-γ signaling promotes Cx3cr1−/−
macrophages towards M2 differentiation in the setting of acute
schistosomiasis, we examined indoleamine 2,3-dioxygenase (IDO)
expression, an immunosuppressive marker relevant to the
functionality of M2 macrophages. Indeed, SEA induced a 1.0-fold
higher IDO expression in Cx3cr1−/− macrophages than in control
macrophages (Fig. 7D). Taken together, these data suggest that loss of
Cx3cr1 enhances IL-4 and IL-10 secretion, which then promotes
STAT6 and PPAR-γ signaling to promote macrophages towards M2
polarization, as manifested by the high levels of IDO expression.
During acute schistosomiasis worm ova released from adult
S. japonicum elicit potent pro-inflammatory responses along with
characteristic granuloma formation, which then causes substantial
injuries to organs such as the liver and intestine where the eggs are
trapped (Hirata et al., 2001). As a result, anti-inflammatory
responses to limit excessive liver injury or intestinal hemorrhage
are necessary to prevent host lethality (Herbert et al., 2008). In
general, acute schistosomiasis is considered a Th1 disease (de Jesus
et al., 2002), and a defect in developing a Th2 response during acute
schistosomiasis is associated with high lethality in mice (Rani et al.,
2012). Given that alternatively activated M2 macrophages possess a
high capacity for secretion of Th2 cytokines, their role in limiting
pro-inflammatory responses and granuloma formation during acute
schistosomiasis has been highly appreciated.
Previous studies have suggested that the trafficking of monocytes
and macrophages from peripheral blood into the sites of
inflammation is a dynamic, multi-step process, and that Cx3cr1
plays a crucial role in the regulation of macrophage trafficking (Shi
and Pamer, 2011). Indeed, Cx3cr1 has been found to promote
atherosclerosis lesion by regulating macrophage accumulation
(Lesnik et al., 2003; Poupel et al., 2013), and blockade of Cx3cr1
attenuates renal pro-inflammatory responses and fibrosis after
Fig. 3. S. japonica infection induces high levels of hepatic Cx3cr1 expression. (A) Western blot analysis of Cx3cr1 expression in the liver lysates 8 weeks
after S. japonica infection. **P<0.01. (B) Co-immunostaining of Cx3cr1 and F4/80 in the liver sections 8 weeks after S. japonica infection. Substantial macrophage
infiltration along with high levels of Cx3cr1 expression was noted after S. japonica infection as manifested by the colocalization of Cx3cr1 and F4/80.
ischemia-reperfusion insult (Furuichi et al., 2006). Based on these
observations, we assessed the role of Cx3cr1 in hepatic granuloma
formation in the setting of acute schistosomiasis by employing
Cx3cr1−/− mice. Remarkably, loss of Cx3cr1 significantly
protected mice from liver injury and functional impairment
mediated by S. japonicum infection, as manifested by reduced
body weight loss, attenuated hepatomegaly and suppressed
granuloma formation along with preserved liver function. Studies
in WT control mice further revealed that S. japonicum infection
induced high levels of hepatic Cx3cr1 expression, and the induced
Cx3cr1 was predominantly expressed by the infiltrating
macrophages. Taken together, our data support the hypothesis that
that inhibition of Cx3cr1 signaling could be an effective approach to
limit pro-inflammatory responses and granuloma formation during
For the first time, we demonstrated that loss of Cx3cr1 causes
macrophages to preferentially polarize to an M2 phenotype during
S. japonicum infection. As discussed earlier, acute schistosomiasis
is characterized by a Th1 response, and indeed, previous studies in
WT mice have demonstrated a significant upregulation of iNOS, a
particular marker for Th1-deviated responses during schistosome
infection (Brunet et al., 1997). Interestingly, Cx3cr1−/− mice
manifested an opposite phenotype, in which significantly higher
levels of Arg-1, a typical marker for Th2 responses, were detected,
whereas iNOS expression was significantly suppressed as
compared to WT control mice. These data provide suggestive
evidence supporting the idea that Cx3cr1 deficiency skews
macrophages preferentially towards M2 polarization during acute
schistosomiasis. To confirm this assumption, we conducted studies
in peritoneal macrophages that were stimulated with extracted
SEA. Indeed, SEA induced a potent Th1 response in WT
macrophages as manifested by the upregulation of iNOS
expression along with enhanced TNF-α and IFN-γ secretion.
However, a typical Th2 response was noted in Cx3cr1−/−
macrophages, as characterized by the high levels of expression of
Arg-1 along with increased IL-4 and IL-10 secretion after SEA
exposure. Notably, flow cytometry analysis of CD206 expression,
a marker associated with M2 polarization, revealed that the
majority of macrophages were positive for CD206 after SEA
stimulation. Taken together, our data demonstrate that Cx3cr1
deficiency leads to a typical switch of the host immune defense
from a conventional Th1 to a typical Th2 response during acute
It has been well demonstrated that control of macrophage
polarization is largely attributed to the function of a small group of
factors including STATs and PPARs (Sajic et al., 2013), and that,
particularly, phosphorylation of STAT6 is a common signaling
factor relevant to M2 polarization of macrophages (Mishra et al.,
Fig. 4. Western blot analysis of hepatic Arg-1 and iNOS expression during acute schistosomiasis. (A) S. japonica infection (8 weeks) did not result in a
perceptible change in the expression of Arg-1 in the liver. (B) S. japonica infection (8 weeks) induced high levels of iNOS expression in the liver. (C) Loss of Cx3xr1
significantly induced Arg-1 expression in the setting of S. japonica infection (8 weeks). (D) Cx3cr1 deficiency significantly attenuated S. japonica-induced iNOS
expression in the liver. Four mice were analyzed for each study group. **P<0.01, ***P<0.001. (E) There was no significant difference in the expression of Arg-1 in
the liver between WT and Cx3cr1−/− mice before S. japonicum infection. (F) No significant difference in terms of hepatic iNOS expression between WT and
Cx3cr1−/− mice was observed before S. japonicum infection.
2011). We thus next examined STAT6 and PPAR-γ signaling
to address the mechanisms by which Cx3cr1 deficiency
skews macrophages towards M2 polarization during acute
schistosomiasis. In accordance with our expectation, SEA
induced a significant increase in STAT6 expression in Cx3cr1−/−
macrophages along with much higher levels of phosphorylated
STAT6 ( p-STAT6) as compared to in WT control macrophages.
Similarly, significantly higher levels of PPAR-γ were detected in
Cx3cr1−/− macrophages than WT control macrophages. We
further noted that SEA stimulation resulted in a 1.0-fold higher
IDO expression in Cx3cr1−/− macrophages than in control
macrophages. Previous studies have demonstrated that IDO
expression in macrophages serves as an important mechanism to
limit the pro-inflammatory response in the setting of tissue and
organ injury or infection (Kim et al., 2009), and defective IDO
production from macrophages results in greater inflammatory
infiltration along with excessive collateral damage to parasitized
organs (Rani et al., 2012). Therefore, these data support the notion
that loss of Cx3cr1 promotes STAT6 and PPAR-γ signaling along
with enhanced IDO expression, which then promotes M2
polarization of macrophages upon acute schistosome infection.
It should be noted that we only investigated the impact of Cx3cr1
deficiency on the polarization of M2 macrophages in the setting of
acute schistosomiasis, therefore, we cannot completely exclude the
possibility that Cx3cr1 deficiency also affects additional pathways.
Furthermore, we only examined pro-inflammatory responses and
hepatic granuloma formation during acute schistosomiasis; the
impact of Cx3cr1 deficiency on hepatic fibrosis in the setting of
chronic schistosomiasis, however, is yet to be elucidated, which
should be a major focus for future studies. Nevertheless, our present
data provide evidence supporting the hypothesis that CX3CR1
could be a viable therapeutic target during schistosomiasis. In fact,
disruption of CX3CR1 signaling has been utilized in
proof-ofconcept preclinical studies (Ishida et al., 2008). The first CX3CR1
antagonist with anti-inflammatory activity both in mice and humans
was recently described (Dorgham et al., 2009). Selective CX3CR1
antagonists could be also beneficial for limiting chronic
inflammatory process during S. japonicum infection.
In summary, we demonstrated that blockade of Cx3cr1 signaling
provides protection for mice against pro-inflammatory responses
and hepatic granuloma formation in the setting of acute
schistosomiasis. Our mechanistic studies revealed that loss of
Cx3cr1 renders macrophages preferentially towards M2
polarization, which involves STAT6 and PPAR-γ signaling along
with enhancement of IDO expression. Taken together, our data
provide evidence suggesting that CX3CR1 could be a viable
therapeutic target in the clinical setting of patients with acute
Fig. 5. Cx3cr1−/− macrophages manifest enhanced Arg-1 and repressed iNOS expression after SEA stimulation. (A) SEA stimulation induced expression
of significantly higher levels of Cx3cr1 in macrophages. (B) SEA induced a 1-fold increase of iNOS expression in macrophages. (C) Addition of SEA significantly
attenuated Arg-1 expression in macrophages. (D) Cx3cr1−/− macrophages manifested a slightly higher, but not a statistically significant, Arg-1 expression as
compared with WT macrophages before SEA stimulation. (E) Loss of Cx3cr1 resulted in a 1.5-fold reduction of SEA-induced iNOS expression in macrophages.
(F) Macrophages deficient in Cx3cr1 manifested a 1-fold higher Arg-1 after SEA stimulation. *P<0.05; **P<0.01.
MATERIALS AND METHODS
C57BL/6 mice and B6 Cx3cr1−/− mice were obtained from the Jackson’s
Laboratory (Bar Harbor, ME, USA). The mice were housed in the SPF
animal facility of Tongji Medical College, Huazhong University of Science
and Technology in microisolator cages supplied with sterile food and water
with a 12-h light and 12-h dark cycle. All animal procedures were performed
in accordance with the National Institute of Health guidelines and were
approved by the Animal Care and Use Committee at Tongji Hospital of
Huazhong University of Science and Technology.
Infection of mice with S. japonicum
Each mouse was infected with 30 cercariae of S. japonicum percutaneously
through a shaved abdomen using the coverslip method as described
previously (Maeda, 1982). Cercariae of S. japonicum originating from
Oncomelania hupensis snails were purchased from the Jiangsu Institute of
Parasite Disease (Wuxi, China). The mice were sacrificed 8 weeks after
infection, and tissues and organs were collected for experimental purposes.
Fifteen mice were included for each study group.
Preparation of soluble egg antigen
Parasitic eggs were isolated from the liver of B6 mice 8 weeks after infection
with cercariae by enzymatic digestion using 0.01% pronase and 0.05%
collagenase (Sigma Chemical Co., St Louis, MO). The eggs were suspended
in 4°C PBS and then homogenized on ice until more than 95% of the eggs
were disrupted. The supernatants were collected and sterilized by passing
through a 0.2-µm filter after ultracentrifugation.
Histological analysis and immunostaining
The collected tissues and organs were first fixed in 4% paraformaldehyde
overnight and then embedded in paraffin. Tissue sections (5 μm) were
prepared using a Leica HM-325 rotary microtome, and then subjected to
H&E staining as previously described (Zhong et al., 2014). For
immunostaining, the sections were first deparaffinized in xylene and
rehydrated in graded alcohol. Nonspecific proteins were blocked with 10%
goat serum or rabbit serum for 30 min. The sections were then probed with a
rat-derived anti-F4/80 antibody (Serotec, Raleigh, NC, USA; 1:100) and a
rabbit-derived anti-CX3CR1 antibody (Abcam, Cambridge, MA; 1:100) at
4°C overnight, followed by incubation with an Alexa-Fluor-546-labeled
anti-rat-IgG and an Alexa-Fluor-594-labeled anti-rabbit-IgG secondary
antibody (Invitrogen, Carlsbad, CA; 1:500) at room temperature for 30 min
as previously reported (Rao et al., 2011). Sections stained with normal IgG
were used as a negative control. Immunofluorescence images were acquired
using a scanning sequential mode to avoid bleed-through effects with a
fluorescence confocal microscope (Carl Zeiss LSM 710, Germany), and
images were processed using the ZEN 2009 software (Carl Zeiss, Germany).
Assessment of hepatic granuloma formation
Hepatic granuloma area was assessed in H&E-stained sections originating
from each group of mice, and 30 granulomas were included for each group of
Fig. 6. Phenotypic analysis of Cx3cr1−/− macrophages after SEA stimulation. (A) Flow cytometry analysis of CD206 expression in F4/80+ CD11b+
macrophages after SEA stimulation. Cx3cr1−/− macrophages manifested a significantly higher proportion of CD206+ cells as compared to WT macrophages.
(B) Comparison of temporal morphological characteristics between WT and Cx3cr1−/− macrophages after SEA stimulation. The locations of the enlarged insets
are indicated by arrows. (C) ELISA analysis of IL-4 and IL-10 secretion into culture supernatant after SEA stimulation. (D) The production of TNF-α and IFN-γ from
macrophages after SEA stimulation. Three replications were conducted for all studies. *P<0.05; **P<0.01; ***P<0.001.
mice. The granuloma size (μm2) was defined by the area containing a single
schistosome egg, and liver sections from 8 mice in each group were randomly
chosen for statistical analysis of granuloma areas. The sections were evaluated
under an Olympus AX-80 microscope, and images were taken using an
Olympus DP 71 camera. Granuloma area was then calculated using the
IMAGE PRO PLUS software package v. 6.0 by tracing the border of
granulomas. An experienced pathologist evaluated all sections in a blinded
fashion, and the severity of inflammatory infiltration was also assessed.
Peritoneal macrophage isolation, culture and treatment
Peritoneal macrophages from both wild-type (WT) B6 and Cx3cr1−/− mice
were isolated as previously reported (Zhong et al., 2010). Briefly, the mice
were intraperitoneally injected with 5 ml sterilized cold RPMI 1640.
Peritoneal macrophages were harvested by washing peritoneal lavage twice
with 5 ml cold RPMI 1640. After lysis of red blood cells, the cells were
incubated for 3 h at 37°C in 35-mm×15-mm tissue culture dishes.
Nonadherent cells were removed by exhaustive washing with 1× PBS. Viability of
macrophages was assessed by the Trypan Blue exclusion method. The
adherent macrophages were next plated at a density of 1×106 cells/ml
followed by stimulation with 1 µg of SEA. The cells and culture supernatants
were collected for experimental purpose 96 h after addition of SEA.
ELISA analysis of cytokine production
The levels of TNF-α, IFN-γ, IL-4 and IL-10 in the serum samples and
culture supernatants were measured with enzyme-linked immunosorbent
assay (ELISA) kits purchased from BD Biosciences (San Jose, CA) using
the established techniques within the laboratory (Han et al., 2008).
Western blot analysis
Liver tissues and cultured macrophages were homogenized in RIPA lysis
buffer (Beyotime, Shanghai, China) using a BBX24 Bullet Blender
homogenizer (Next Advance Inc., Averill Park, NY) according to the
manufacturer’s instruction. The lysates were separated by 10% SDS-PAGE
and then transferred onto polyvinylidene difluoride (PVDF) membranes.
Western blot analysis was carried out as reported previously by probing the
blots with the indicated primary antibody followed by a horseradish
peroxidase (HRP)-conjugated secondary antibody (Yang et al., 2013). The
reactive bands were visualized using an ECL Plus Western Blotting
kit (PIERCE, Rockford, IL) according to the manufacturer’s instructions.
Primary antibodies against CX3CR1 (1:1000), indoleamine 2, 3-dioxygenase
(IDO, 1:1000), STAT6 (1:500), PPAR-γ (1:500), and β-actin (1:500) were
obtained from Abcam (Cambridge, MA), whereas antibodies against iNOS
(1:250) and Arg-1 (1:4000) were purchased from BD Pharmingen
(Carlsbad, CA). The intensity of each reactive band was analyzed using
the densitometry plugin ImageJ software (http://rsb.info.nih.gov/ij/).
Flow cytometry analysis
Surface marker expression on macrophages 96 h after SEA stimulation
was determined by flow cytometry as previously reported (Han et al., 2008).
Briefly, macrophages were first washed twice with FACS medium
Fig. 7. Loss of Cx3cr1 enhances STAT6 and PPAR-γ signaling. (A) Western blot analysis of total STAT6 in macrophage lysates after SEA stimulation. (B)
Western blot results for phosphorylated STAT6 ( p-STAT6). (C) Western blot results for PPAR-γ. (D) Western blot analysis of IDO in macrophages after SEA
stimulation. *P<0.05; **P<0.01.
(2% heat-inactivated FCS in PBS), followed by incubation with
phycoerythrin-labeled F4/80 (eBioscience, Danvers, MA), APC-labeled
CD11b and FITC-labeled CD206 (BD Pharmingen, Carlsbad, CA) at 4°C
for 30 min. After washes, the cells were subjected to flow cytometry
analysis, and all data were analyzed using the FACSCalibur and FlowJo
version X software.
All data are shown as mean±s.e.m. All in vitro experiments were conducted
with three independent replications. Graphpad Prism 5 was used for
statistical analysis using Student’s t-test or one-way or two-way ANOVA
and Bonferroni’s post hoc test where appropriate. In all cases, P<0.05 was
considered statistically significant.
L.R. contributed to the acquisition of data; B.-X.R. and Q.Y. contributed to the study
design; J.C. and P.Y. contributed to the statistical analysis; H.N., S.Z., Q.Z. and X.Y.
contributed to the analysis and interpretation of data; M.K., P.K., W.G. and J.-F.X.
contributed to the critical revision of the manuscript for important intellectual content;
C.-Y.W. contributed to the drafting of the manuscript and obtained funding; F.Y., F.X.
and Q.G. contributed to the technical support.
This work was supported by grants from the National Natural Science Foundation of
China [grant numbers 81130014, 81471046, 81470988 and 81271872]; the Chinese
Ministry of Education for Innovative Research Team [grant number IRT_14R20]; the
European Foundation for the Study of Diabetes (EFSD)/Chinese Diabetes Society
(CDC)/Lilly Program for Collaborative Diabetes Research between China and Europe;
and the Innovative Funding for Translational Research from Tongji Hospital.
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