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Decidual macrophage M1 polarization contributes to adverse pregnancy induced by Toxoplasma gondii PRU strain infection
Accepted Manuscript Decidual macrophage M1 polarization contributes to adverse pregnancy induced by Toxoplasma gondii PRU strain infection Xianbing Liu, Mengqi Jiang, Liqin Ren, Aihong Zhang, Mingdong Zhao, Haixia Zhang, Yuzhu Jiang, Xuemei Hu PII:
S0882-4010(18)30971-9
DOI:
10.1016/j.micpath.2018.08.043
Reference:
YMPAT 3128
To appear in:
Microbial Pathogenesis
Received Date: 27 May 2018 Revised Date:
24 July 2018
Accepted Date: 20 August 2018
Please cite this article as: Liu X, Jiang M, Ren L, Zhang A, Zhao M, Zhang H, Jiang Y, Hu X, Decidual macrophage M1 polarization contributes to adverse pregnancy induced by Toxoplasma gondii PRU strain infection, Microbial Pathogenesis (2018), doi: 10.1016/j.micpath.2018.08.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Decidual macrophage M1 polarization contributes to adverse
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pregnancy induced by Toxoplasma gondii PRU strain infection
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Xianbing Liua,1, Mengqi Jiangb,1, Liqin Rena,1, Aihong Zhangc, Mingdong Zhaod,
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Haixia Zhanga, Yuzhu Jianga and Xuemei Hua,*
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a
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Medical University, Yantai, China
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b
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Medical University, Binzhou, China
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Department of Immunology, Medicine and Pharmacy Research Center, Binzhou
Department of Gynecology and Obstetrics, Binzhou Affiliated Hospital of Binzhou
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School Hospital of Binzhou Medical University, Yantai, China
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Department of Radiology, Binzhou Affiliated Hospital of Binzhou Medical University,
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Binzhou, China
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Running Title： ：T. gondii infection causes M1 polarization in the decidua
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*Corresponding author. Department of Immunology, Medicine and Pharmacy
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Research Center, Binzhou Medical University, No.346 Guanhai Road, Yantai 264003,
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Shandong, China.
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E-mail addresses: [email protected] (X. Hu)
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These authors contributed equally to this work.
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ABSTRACT Recent evidence indicates that macrophages at the maternal–fetal
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interface adapt to a phenotype characterized by alternative activation (M2
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polarization) and exhibit immunosuppressive functions that favor the
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maintenance of pregnancy. The bias of M2 decidual macrophages toward
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M1 has been clinically linked to pregnancy-related complications, such as
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preeclampsia, and preterm delivery. The aim of this study was to
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investigate the effect of Toxoplasma gondii PRU strain infection on the
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bias of decidual macrophage polarization and its contribution to adverse
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pregnancy outcomes. A mouse model with adverse pregnancy outcome
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was established by infection with T. gondii PRU strain and the expression
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levels of functional molecules in decidual macrophages of mice were
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measured. The results showed that T. gondii infection caused seriously
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adverse pregnancy outcome in mice. The placentae of infected mice
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showed obvious congestion and inflammatory cell infiltration. The
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expression of CD206, MHC-II, and arginase-1 considered as M2 markers
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was decreased in decidual macrophages after T. gondii infection, whereas
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the expression of CD80, CD86, iNOS, and cytokines TNF-α and IL-12
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considered as M1 markers was increased. Furthermore, iNOS-positive
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expression was observed in the decidua basalis of infected mice. Our
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results indicated that T. gondii infection was responsible for the bias of
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M2
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immunosuppressive microenvironment at the maternal–fetal interface and
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contributes to adverse pregnancy outcomes.
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decidual
macrophages
toward
M1,
which
changes
the
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Keywords: Toxoplasma gondii PRU strain; decidual macrophages;
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polarization; adverse pregnancy outcomes
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1. Introduction The protozoan parasite Toxoplasma gondii (T. gondii), an obligate
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intracellular eukaryotic parasite of the phylum apicomplexa, may cause
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toxoplasmosis in many warm-blooded animals, including humans.
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Approximately one-third of the human population has been exposed
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to this parasite worldwide [1]. Although T. gondii infection may be
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usually
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life-threatening in immunocompromised patients (e.g., AIDS patients or
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those receiving immunosuppressive drug therapy for malignancies or
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organ transplantation) [2]. Furthermore, if a primary infection with T.
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gondii occurs during pregnancy, particularly during the first trimester, this
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may result in adverse pregnancy outcomes, such as stillbirth, miscarriage,
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preterm labor, or congenital toxoplasmosis in the surviving offspring [3].
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Furthermore, of congenitally infected babies without symptoms at birth,
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three-quarters will later develop severe mental retardation and/or hearing
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defects and as many as 90% will suffer eye problems as they grow older,
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which results in a huge drain on the public health system [4]. Previous
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studies have demonstrated that T. gondii infection-induced immune
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dysfunction at the maternal–fetal interface contributed to adverse
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pregnancy outcomes [5–7]. However, the underlying immune mechanism
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is not yet fully elucidated.
immunocompetent
individuals,
it
is
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asymptomatic
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During pregnancy, macrophages represent a major leukocyte subset
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and comprise approximately 20%–30% of all decidual leukocytes [8].
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Macrophages can be phenotypically polarized by the microenvironment
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in which they reside and can be categorized into two groups: classically
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activated macrophages (M1 macrophages) and alternatively activated
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macrophages (M2 macrophages) [9]. M1 macrophages are characterized
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by the expression of CD16/32, CD80, CD86, and inducible nitric oxide
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synthase (iNOS, also named NOS2) and the production of Th1-type 3
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cytokines. M2 macrophages are characterized by the upregulation of
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mannose receptor (CD206) and arginase-1, production of Th2-type
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cytokines, and demonstration of anti-inflammatory activity [10,11].
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Accumulating evidence indicates that decidual macrophages have an
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immunosuppressive, M2-like phenotype [12–14]. This phenotype of
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decidual macrophages may contribute to both remodeling of the
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endometrium and the tolerant milieu required for fetal acceptance.
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Aberrant phenotypes of decidual macrophages have been reported to
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associate with pathological pregnancy in human [15,16]. Decidual
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M2-like macrophages were reduced in preterm pregnancies compared
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with term pregnancies and decidual macrophages expressed high levels of
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tumor necrosis factor (TNF) and interleukin (IL)-12 during spontaneous
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preterm labor [15]. In the deciduas of patients with unexplained recurrent
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miscarriages, macrophage expression of CD80 and CD86 was higher than
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that in normal control women during early pregnancy; whereas the
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expression of IL-10 was lower [16]. In the abortion-prone (female CBA ×
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male DBA) murine mating model, the polarization patterns of placental
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macrophages were also characterized mainly by M1 phenotypes [17].
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M1-activated macrophages may damage the conceptus via production of
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nitric oxide and TNF-α [18].
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Most T. gondii strains isolated from humans and animals in North
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America and Europe have been grouped into three predominant clonal
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lineages (types I, II and III). These strains differ in virulence in mice and
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likely cause different sequelae in humans [19]. In our previous study, we
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have reported that the infection of T. gondii RH strain (type I) can
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down-regulated LILRB4 in decidual macrophages and contribute their
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functional polarization during abnormal pregnancy [20]. Nevertheless,
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the majority of human toxoplasmosis is due to infection by type II strains
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which are the most prevalent in nature [21,22], and the effect of infection
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with T. gondii type II strains on the polarization of decidual macrophages
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need further research. In the present study, an adverse pregnancy outcome
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mouse model induced by T. gondii PRU strain (Type II) infection in the
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first trimester was used to explore the immunopathology mechanism
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related to decidual macrophages.
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2. Materials and methods
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2.1. Mice and mating
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C57BL/6 mice (8–10 weeks old) were purchased from Beijing Weitong
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Lihua Experimental Animals Technical Co., Ltd. All mice were housed
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(five mice/cage) at 20°C–24°C in a specific-pathogen-free animal room
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on a 12-h light–dark cycle, with free access to food and water. Following
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overnight cohabitation with males at a ratio of 2:1, females with a vaginal
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plug, considered as gestational day (gd) 0, were segregated and
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randomized to the infection group or control group. The following animal
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experiments were approved by the Institutional Animal Experimental
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Ethics Committee of Binzhou Medical University.
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2.2. Toxoplasma gondii
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T. gondii PRU strain (Type II) was kindly provided by Professor
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Jilong Shen, Department of Pathogen Biology, Anhui Medical University,
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Anhui, China and was used to infect the mice. Cysts of T. gondii PRU
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strain were harvested from the brains of Kunming mice chronically
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infected 30–45 days earlier with approximately 20 cysts.
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2.3. Infection and pregnancy outcomes
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Pregnant mice in the infection group were perorally inoculated with
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15 cysts in 300 µl phosphate-buffered saline (PBS) on gd 4, whereas
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control mice were treated with 300 µl PBS solution. On gd 14, the mice 5
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were euthanatized by cervical dislocation, the uteri were removed, and
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the total numbers of normal and abnormal embryos were recorded. The
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abnormal embryos were identified by their small size, hemorrhagic and
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necrotic appearance, or complete resorption compared with normal
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embryos. The adverse pregnancy rate was calculated as the ratio of
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abnormal embryo numbers to total embryo numbers.
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2.4. Histopathological and immunohistochemical analysis
Following euthanasia on gd 14, randomly selected uteri and
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placentae of pregnant mice were fixed in 4% paraformaldehyde/PBS and
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routinely processed for paraffin embedding and sectioning. Tissue
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sections were cut at 4 µm and stained with hematoxylin and eosin (H & E;
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Boster Biotechnology, Wuhan, China) for histopathological analysis.
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Sections were examined on a photomicroscope.
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For immunohistochemistry assay, tissue sections were deparaffinized
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and rehydrated. Heat-induced antigen retrieval was performed with 0.01
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M citrate buffer (pH 6.0; Boster Biotechnology) at 95°C for 20 min.
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Endogenous peroxidase was blocked by 3% hydrogen peroxide in
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methanol. Sections were incubated with 2% bovine serum albumin for 30
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min at 37°C to block non-specific binding sites and with anti-mouse
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iNOS polyclonal antibody (mAb) (1:50 dilution; Proteintech, Wuhan,
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China) overnight at 4°C. Immunohistochemical staining was detected
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using
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tetrahydrochloride (both from Boster Biotechnology). The sections were
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counterstained with Mayer’s hematoxylin and examined under a light
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microscope.
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a
SABC
kit
and
developed
with
diaminobenzidine
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2.5. Cell preparation and flow cytometry Single-cell suspensions were prepared from placental and uterine 6
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tissues by dissecting the tissues into 1–3-mm pieces. The pieces were
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digested with 0.15% trypsin and 25 U/ml DNase I (both from
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Sigma-Aldrich, St. Louis, MO, USA) in RPMI 1640 (Hyclone, Logan,
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UT, USA) at 37°C for 45 min under gentle stirring. Subsequently, the
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samples were ground and filtered through a 75-µm pore size sieve.
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Mononuclear cells were isolated by Ficoll–Hypaque density gradient
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centrifugation.
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Fc receptors of cells were blocked with 0.5 µg of anti-CD16/32 mAb
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(BD Pharmingen, San Diego, CA, USA) in 100 µl FACS buffer (3% FBS
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and 0.1% sodium azide in PBS, pH 7.2) for 20 min at 4°C. The following
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fluorophore-conjugated anti-mouse mAbs were used: anti-F4/80-PE-Cy7,
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anti-CD206-PE, anti-MHC-II (I-A/I-E)-FITC (all from BioLegend, San
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Diego, CA, USA), anti-CD80-PE, and anti-CD86-PE mAbs (both from
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BD Pharmingen). The blocked cells were incubated with mAbs at 4°C in
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darkness for 30 min and subsequently washed twice with PBS. For
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intracellular cytokine staining, cells were cultured with Leukocyte
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Activation Cocktail and BD GolgiPlug (both from BD Biosciences,
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Franklin Lakes, NJ, USA) for 4–6 h at 37°C, according to the
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manufacturer’s instructions. After staining of F4/80, the cells were fixated
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and permeabilized in 1× Fix/Perm buffer (Invitrogen, Carlsbad, CA, USA)
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for 30 min. Subsequently, the cells were stained with anti-mouse
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TNF-α-FITC (BioLegend), anti-IL-12p70-PE, and IL-10-PE (both from
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BD Pharmingen) in darkness for 30 min at 4°C. In parallel, the
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corresponding isotypes were used as controls. Flow cytometric data were
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acquired using a BD FACSCantoTM II flow cytometer and analyzed using
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the FACSDiva software (BD Biosciences).
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2.6. Enzyme-linked immunosorbent assay (ELISA) Homogenates of placental tissues were prepared in PBS with a 7
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homogenizer and then centrifuged at 12,000 g for 30 min at 4°C.
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Supernatants were collected and stored at −80°C until analysis. TNF-α,
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IL-12p70, and IL-10 were measured using ELISA kits (Lengton
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Bioscience,
202
instructions. The concentration of cytokines in the placental tissue
203
homogenates was calculated from a standard curve of each murine
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recombinant cytokine. Each measurement was performed in triplicate.
China),
according
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2.7. Magnetic cell sorting and Western blotting
the
manufacturer’s
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The mononuclear cells of mice uteri and placentae were acquired as
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described above, and F4/80 positive cells were sorted with anti-F4/80
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MicroBeads (Miltenyi Biotec, Bergish Gladbach, Germany), according to
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the manufacturer’s instructions. In all cases, the purity of sorted cells was
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at least 80%, as determined by flow cytometry.
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Protein extraction from sorted macrophages was performed in a lysis
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buffer (lysis buffer/PMSF = 16:1) (Beyotime, Shanghai, China). The cell
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lysates were subsequently centrifuged at 12,000 g for 15 min at 4°C. The
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denatured protein samples (20 µg protein/lane) were separated by
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standard SDS-PAGE with 10% separating gel and transferred onto a
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PVDF membrane (Merck Millipore, Billerica, MA, USA). After blocking
218
with 5% skim milk, the membrane was probed using purified anti-mouse
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iNOS and arginase-1 mAbs (both 1:2,000 dilution; BD Pharmingen).
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β-actin was stained with antibody (1:5,000 dilution; Santa Cruz, Dallas,
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TX, USA) as control. The samples were labeled using horseradish
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peroxidase-labeled secondary antibodies (1:10,000 dilution; Boster), and
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chemiluminescence was evaluated using an enhanced chemiluminescence
224
kit (Roche Diagnostics, Indianapolis, IN, USA). The results were
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analyzed using the Image J 1.46 software (NIH image, USA).
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2.8. Data analysis All data are presented as means ± standard deviation (SD). Statistical
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analyses were performed using the SPSS 16.0 statistical software package
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(SPSS, Inc., Chicago, IL, USA). The unpaired Student’s t-test was used to
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evaluate differences between the two groups. P values of <0.05 or <0.01
232
were considered as significant or very significant, respectively.
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3. Results
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3.1. Establishing an animal model with T. gondii infection-induced
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adverse pregnancy outcome
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In the present study, pregnancy outcomes were observed on gd 14
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after 10 days of T. gondii infection. The pregnant mice infected with T.
239
gondii PRU strain showed malaise, extrados, and erected fur, whereas the
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control mice did not show discernible abnormal symptoms. Adverse
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pregnancy outcomes occurred following T. gondii infection (Fig. 1A and
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1B).
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H & E staining of paraffin sections showed that infected mice had
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obvious congestion in the placenta and a larger intervillous space (Fig. 1F
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and 1G) compared with the control mice (Fig. 1C and 1D). Furthermore,
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there were numerous inflammatory cells with round nuclei existing in the
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placental villi of the infected pregnant mice (Fig. 1H), whereas few
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inflammatory cells were found in the control samples (Fig. 1E).
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3.2. T. gondii infection upregulated the expression of M1 functional
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molecules in decidual macrophages and downregulated the expression of
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M2
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The expression of M1 (CD80 and CD86) and M2 (CD206 and
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MHC-II) was analyzed using flow cytometry to investigate the effect of T.
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gondii infection on the surface functional molecules of decidual 9
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macrophages. The expression of CD80 and CD86 was upregulated,
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whereas the expression of CD206 and MHC-II was downregulated in the
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infection group compared with the control group (Fig. 2).
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3.3. T. gondii infection increased the expression of iNOS and decreased
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arginase-1 in decidual macrophages
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To determine the effect of T. gondii infection on L-arginine
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metabolism of decidual macrophages, the expression of iNOS and
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arginase-1 in the sorted decidual macrophages was analyzed using
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Western blotting. Compared with the control group, iNOS expression in
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the infection group was obviously increased, whereas arginase-1
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expression was significantly decreased (Fig. 3A and 3B).
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Immunohistochemical staining was also employed to determine the
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expression of iNOS in the placenta and uterus. In our results, iNOS
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expression was undetected in the control mice (Fig. 3C and 3D).
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Following T. gondii infection, iNOS-positive expression was observed in
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the interface of uterus and placenta, in which macrophages reside (Fig.
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3E and 3F).
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3.4. T. gondii infection altered the cytokine secretion of decidual
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macrophages
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Flow cytometry was used to investigate the effect of T. gondii
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infection on cytokine secretion of decidual macrophages. Following T.
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gondii infection, levels of TNF-α, IL-12, and IL-10 in decidual
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macrophages were significantly increased. However, the ratios of
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TNF-α/IL-10 and IL-12/IL-10 in the infection group were higher than
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those in the control group. These results suggest that T. gondii infection
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altered the secretion pattern of cytokines related with M1/M2 polarization
284
of decidual macrophages (Fig. 4A–4C). 10
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In addition, cytokines in the supernatant of placental homogenates
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were also measured using ELISA. Levels of TNF-α, IL-12, and IL-10 in
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the infection group were higher than those in the control group. Moreover,
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the ratios of TNF-α/IL-10 and IL-12/IL-10 in the infection group were
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also higher than those in the control group. The change in cytokine
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secretion pattern in placentae was consistent with that observed in the
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decidual macrophages (Fig. 4D and 4E).
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4. Discussion
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Maternal immune cells, including macrophages, natural killer cells,
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dendritic cells, and regulatory T cells are regulated at the maternal–fetal
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interface to tolerate the expression of paternal antigens by the
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semi-allogenic fetus without immune rejection. Dysfunction of these
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immune cells or dysregulation of the maternal–fetal immune tolerance
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may lead to miscarriage, preterm delivery, or severe complications, such
300
as preeclampsia and fetal growth restriction [23]. T. gondii is one of the
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TORCHES (toxoplasmosis, rubella, cytomegalovirus, herpes simplex,
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and syphilis) pathogens that cause severe adverse pregnancy outcomes
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when contacted during pregnancy [24]. Our previous studies have
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demonstrated that T. gondii infection may cause aberrant activation of
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natural killer cells and dendritic cells in the deciduas and lead to
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dysfunction of regulatory T cells, which are closely associated with T.
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gondii infection-induced adverse pregnancy outcomes [5,6,25]. In the
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present study, we investigated the effect of T. gondii PRU strain infection
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on decidual macrophage polarization and aimed at providing new insight
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on the immune mechanism linked to adverse pregnancy outcomes
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induced by this pathogen.
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Macrophages are vital immune cells in the deciduas and
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predominantly display an M2 phenotype. Decidual macrophages typically 11
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express lower levels of M1 functional molecules (CD80 and CD86) and
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higher levels of M2 functional molecules (CD206 and MHC-II) to sustain
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maternal–fetal immune tolerance during normal pregnancy [26,27]. On
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the contrary, abnormal expression of these molecules has been associated
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with the pathology of pregnancy. For example, upregulated CD80 and
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CD86 expression in macrophages has been reported in the deciduas of
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patients with unexplained recurrent miscarriage compared with normal
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control women during early pregnancy [16]. Moreover, a lower
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CD206/CD68 mRNA expression ratio in first-trimester decidual tissue
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was found in pregnancies complicated with hypertension [28].
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Furthermore, it has reported that the combined use of anti-CD80 and
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anti-CD86 mAbs can induce maternal tolerance of the fetus in the
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abortion-prone CBA/J females mated with DBA/2 males [17]. However,
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the effect of T. gondii infection on the expression of M1- and M2-related
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functional molecules and polarization of decidual macrophages during
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pregnancy remains unexplored. The present study employed a mouse
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pregnancy model with T. gondii PRU strain infection to investigate the
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effect of infection on the expression of membrane functional molecules of
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decidual macrophages. Our results showed that T. gondii infection
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upregulated the expression of M1 functional molecules CD80 and CD86
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of decidual macrophages. In contrast, the infection downregulated the
335
expression of M2 functional molecules CD206 and MHC-II. This T.
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gondii infection-induced change in the decidual macrophage phenotype
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enhanced the immune activity of the decidual macrophages, weakened
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the maternal–fetal tolerance, and may have contributed to the occurrence
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of adverse pregnancy outcomes.
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At the most fundamental level, M1/M2 polarity of macrophages is
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driven by arginine metabolism mediated by iNOS and arginase-1 [11].
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M1 macrophages are characterized by the expression of iNOS and the 12
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production of NO, an important effector for their microbicidal activity
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[29]. However, a high level of iNOS expression and NO production may
345
induce immunopathology due to its autotoxic effect [30]. Excess iNOS
346
expression may suppress placental vascular development, which is
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deleterious to the developing fetus [31]. It has reported that the
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expression of iNOS in human placentae was not detected in normal
349
pregnancy [32]. Nevertheless, our results suggested that the expression of
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iNOS in decidual macrophages can be induced by T. gondii infection. The
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T. gondii-induced high expression of iNOS in decidual macrophages may
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enhance their cytotoxic effect and consequently contribute to adverse
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pregnancy outcomes. On the contrary, M2 macrophages do not produce
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NO and express high levels of arginase-1, which catalyzes the production
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of polyamines [31]. Arginase-1 antagonizes iNOS activity in arginine
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metabolism by competing for arginine and directly inhibits iNOS [33].
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Arginase-1 enhances the bioavailability of L-arginine and promotes
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polyamine synthesis, which is involved in immunosuppression and
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placental development during pregnancy [34,35]. In the present study, T.
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gondii infection significantly decreased arginase-1 expression in decidual
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macrophages. This result indicated that the downregulated arginase-1
362
expression by T. gondii infection may impair its antagonism against iNOS
363
activity and the maternal–fetal immune tolerance. Thus, T. gondii
364
infection skewed decidual macrophages toward M1 by inducing iNOS
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synthesis and away from M2 decidual macrophages by downregulating
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arginase-1 expression. Finally, the bias of M2 decidual macrophages
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toward M1 may contribute to the occurrence of adverse pregnancy
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outcomes caused by T. gondii infection.
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Decidual macrophages also showed an M2 polarized cytokine
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secretion pattern with abundant production of IL-10 [36]. IL-10 is a
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typical anti-inflammatory cytokine, playing a vital role in the maternal 13
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immune tolerance of an allogeneic fetus [37]. IL-10 deficiency leads to
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fetal resorption and preterm birth in pregnant mice when challenged with
374
low doses of LPS or CpG [38,39]. Of note, administration of recombinant
375
IL-10 reverses or alleviates symptoms of adverse pregnancy outcomes in
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animal models [37]. In a previous study, we showed that treatment with
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recombinant IL-10 also alleviated adverse pregnancy outcome induced by
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T. gondii infection in mice [40]. Decidual macrophages also produce
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pro-inflammatory cytokines, such as TNF-α and IL-12, in normal
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pregnancy [41]. But increase of these cytokines, especially TNF-α, is
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associated with several obstetric disorders, such as preeclampsia, fetal
382
growth restriction, preterm labor, and spontaneous and recurrent abortion
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[42].
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macrophages directly induce trophoblast cell apoptosis and exacerbate
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adverse outcomes during pregnancy [43]. In this study, levels of IL-10,
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TNF-α, and IL-12 production in decidual macrophages were determined
387
by flow cytometry. We found that the expression levels of IL-10, IL-12,
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and TNF-α in decidual macrophages were all increased following T.
389
gondii infection. However, TNF-α and IL-12 levels were higher
390
compared with IL-10 levels based on TNF-α/IL-10 and IL-12/IL-10 ratio
391
analysis. Furthermore, measurement of cytokines in the supernatant of
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placental homogenates through ELISA yielded consistent findings with
393
those of cytokines secreted by decidual macrophages. Thus, T. gondii
394
infection mainly increased pro-inflammatory cytokines TNF-α and IL-12,
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which
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implantation sites and further damage the developing embryo. The
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imbalance of M1 and M2 cytokine production in decidual macrophages
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may play an important role in adverse pregnancy outcomes induced by T.
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gondii infection.
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cytokines
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apoptosis/necrosis
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Taken together, our data indicated that T. gondii infection caused the 14
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bias of M2-like decidual macrophages toward M1 through an increase in
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the expression levels of CD80, CD86, iNOS, TNF-α, and IL-12 and a
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decrease in CD206, MHC-II, and arginase-1 levels. The skewing of
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decidual macrophage polarization at the maternal–fetal interface may
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result in weakening of the immunosuppressive microenvironment,
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thereby contributing to adverse pregnancy outcomes induced by T. gondii
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infection.
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The authors declare that they have no competing interests.
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Conflict of interest
Acknowledgments
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This work was supported in part by grants from the National Natural
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Science Foundation of China (81401687, 81672049) and Taishan Scholar
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Foundation.
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Figure legends
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Fig. 1. Adverse pregnancy outcomes caused by T. gondii infection. (A)
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Representative images of mice and uteri from T. gondii-infected mice and
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controls are shown. Noticeable adverse pregnancy outcomes occurred in
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mice infected with T. gondii PRU stain compared with the control mice.
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(B) Following 10 days of infection, the adverse pregnancy rate was
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calculated as the ratio of abnormal embryo numbers to total embryo
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numbers (**P < 0.01). (C–H) H & E staining of paraffin sections from
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control and infected mice on gd 14. (C) The control mice showed normal
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aspects of the uterus (U) and placenta (P). (F) Infected mice showed
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noticeable hyperemia in the placenta. (D, G) The images also showed
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different hyperemia in the placentae of infected and control mice. (E) The
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placentae of control mice showed little inflammatory cell infiltration in
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the placental villi. (H) The placentae of infected mice showed greater
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inflammatory cell infiltration (arrows) in the placental villi.
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Fig. 2. T. gondii infection altered the expression of M1 and M2
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functional molecules in decidual macrophages. Flow cytometry was
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used to measure the expression levels of CD80 (A), CD86 (B), CD206 (C)
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and MHC-II (D), and anti-F4/80 mAb was used to gate decidual
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macrophages. Data are expressed as mean ± SD obtained from six mice
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in each group (**P < 0.01).
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Fig. 3. T. gondii infection affected the expression of iNOS and
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arginase-1 in decidual macrophages. (A) The expression of iNOS and
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arginase-1 in decidual macrophages was analyzed by Western blotting. (B)
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The relative expression of iNOS and arginase-1 is shown. Data are
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represented as mean ± SD of three independent experiments (*P < 0.05,
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**P < 0.01). (C–F) The expression of iNOS in the placenta (P) and uterus 21
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(U) was also analyzed by immunohistochemical staining. iNOS-positive
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cells in tissues were identified by brown staining. Noticeable iNOS
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expression was observed at the interface between uterus and placenta in
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the T. gondii-infected mice (E, F), whereas iNOS expression was
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undetectable in the control mice (C, D). The figure is representative of
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five independent experiments.
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Fig. 4. The secretion pattern of cytokines in decidual macrophages
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was altered following T. gondii infection. (A) The expression of
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cytokine TNF-α, IL-12, and IL-10 was assessed by flow cytometry. Cells
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are gated on F4/80-positive cells, and the scatter diagrams of cytokines
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are shown. (B) The positive rates of TNF-α, IL-12, and IL-10 expressed
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in decidual macrophages were counted. Data are presented as mean ± SD
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obtained from six mice in each group. (C) The ratios of TNF-α/IL-10 and
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IL-12/IL-10 in decidual macrophages are shown. (D) Levels of TNF-α,
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IL-12, and IL-10 in supernatants of placental homogenates were
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determined using ELISA. Each measurement was performed in triplicate.
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Data are represented as mean ± SD obtained from six mice in each group.
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(E) The ratios of TNF-α/IL-10 and IL-12/IL-10 in supernatants of
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placental homogenates are also shown. *P < 0.05, **P < 0.01.
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Highlights
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T. gondii infection changes functional molecule expression in decidual macrophages. T. gondii infection affects iNOS and arginase-1 expression in decidual macrophages. T. gondii infection alters the pattern of cytokine secretion in decidual macrophages.
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T. gondii infection causes M1 polarization of decidual macrophages.
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The bias of M1/M2 polarization contributes to adverse pregnancy outcomes.
S0882-4010(18)30971-9
DOI:
10.1016/j.micpath.2018.08.043
Reference:
YMPAT 3128
To appear in:
Microbial Pathogenesis
Received Date: 27 May 2018 Revised Date:
24 July 2018
Accepted Date: 20 August 2018
Please cite this article as: Liu X, Jiang M, Ren L, Zhang A, Zhao M, Zhang H, Jiang Y, Hu X, Decidual macrophage M1 polarization contributes to adverse pregnancy induced by Toxoplasma gondii PRU strain infection, Microbial Pathogenesis (2018), doi: 10.1016/j.micpath.2018.08.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Decidual macrophage M1 polarization contributes to adverse
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pregnancy induced by Toxoplasma gondii PRU strain infection
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Xianbing Liua,1, Mengqi Jiangb,1, Liqin Rena,1, Aihong Zhangc, Mingdong Zhaod,
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Haixia Zhanga, Yuzhu Jianga and Xuemei Hua,*
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a
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Medical University, Yantai, China
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b
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Medical University, Binzhou, China
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Department of Immunology, Medicine and Pharmacy Research Center, Binzhou
Department of Gynecology and Obstetrics, Binzhou Affiliated Hospital of Binzhou
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School Hospital of Binzhou Medical University, Yantai, China
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d
Department of Radiology, Binzhou Affiliated Hospital of Binzhou Medical University,
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Binzhou, China
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Running Title： ：T. gondii infection causes M1 polarization in the decidua
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*Corresponding author. Department of Immunology, Medicine and Pharmacy
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Research Center, Binzhou Medical University, No.346 Guanhai Road, Yantai 264003,
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Shandong, China.
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E-mail addresses: [email protected] (X. Hu)
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These authors contributed equally to this work.
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ABSTRACT Recent evidence indicates that macrophages at the maternal–fetal
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interface adapt to a phenotype characterized by alternative activation (M2
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polarization) and exhibit immunosuppressive functions that favor the
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maintenance of pregnancy. The bias of M2 decidual macrophages toward
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M1 has been clinically linked to pregnancy-related complications, such as
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preeclampsia, and preterm delivery. The aim of this study was to
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investigate the effect of Toxoplasma gondii PRU strain infection on the
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bias of decidual macrophage polarization and its contribution to adverse
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pregnancy outcomes. A mouse model with adverse pregnancy outcome
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was established by infection with T. gondii PRU strain and the expression
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levels of functional molecules in decidual macrophages of mice were
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measured. The results showed that T. gondii infection caused seriously
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adverse pregnancy outcome in mice. The placentae of infected mice
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showed obvious congestion and inflammatory cell infiltration. The
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expression of CD206, MHC-II, and arginase-1 considered as M2 markers
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was decreased in decidual macrophages after T. gondii infection, whereas
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the expression of CD80, CD86, iNOS, and cytokines TNF-α and IL-12
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considered as M1 markers was increased. Furthermore, iNOS-positive
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expression was observed in the decidua basalis of infected mice. Our
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results indicated that T. gondii infection was responsible for the bias of
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M2
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immunosuppressive microenvironment at the maternal–fetal interface and
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contributes to adverse pregnancy outcomes.
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decidual
macrophages
toward
M1,
which
changes
the
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Keywords: Toxoplasma gondii PRU strain; decidual macrophages;
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polarization; adverse pregnancy outcomes
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1. Introduction The protozoan parasite Toxoplasma gondii (T. gondii), an obligate
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intracellular eukaryotic parasite of the phylum apicomplexa, may cause
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toxoplasmosis in many warm-blooded animals, including humans.
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Approximately one-third of the human population has been exposed
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to this parasite worldwide [1]. Although T. gondii infection may be
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usually
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life-threatening in immunocompromised patients (e.g., AIDS patients or
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those receiving immunosuppressive drug therapy for malignancies or
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organ transplantation) [2]. Furthermore, if a primary infection with T.
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gondii occurs during pregnancy, particularly during the first trimester, this
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may result in adverse pregnancy outcomes, such as stillbirth, miscarriage,
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preterm labor, or congenital toxoplasmosis in the surviving offspring [3].
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Furthermore, of congenitally infected babies without symptoms at birth,
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three-quarters will later develop severe mental retardation and/or hearing
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defects and as many as 90% will suffer eye problems as they grow older,
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which results in a huge drain on the public health system [4]. Previous
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studies have demonstrated that T. gondii infection-induced immune
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dysfunction at the maternal–fetal interface contributed to adverse
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pregnancy outcomes [5–7]. However, the underlying immune mechanism
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is not yet fully elucidated.
immunocompetent
individuals,
it
is
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asymptomatic
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During pregnancy, macrophages represent a major leukocyte subset
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and comprise approximately 20%–30% of all decidual leukocytes [8].
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Macrophages can be phenotypically polarized by the microenvironment
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in which they reside and can be categorized into two groups: classically
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activated macrophages (M1 macrophages) and alternatively activated
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macrophages (M2 macrophages) [9]. M1 macrophages are characterized
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by the expression of CD16/32, CD80, CD86, and inducible nitric oxide
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synthase (iNOS, also named NOS2) and the production of Th1-type 3
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cytokines. M2 macrophages are characterized by the upregulation of
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mannose receptor (CD206) and arginase-1, production of Th2-type
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cytokines, and demonstration of anti-inflammatory activity [10,11].
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Accumulating evidence indicates that decidual macrophages have an
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immunosuppressive, M2-like phenotype [12–14]. This phenotype of
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decidual macrophages may contribute to both remodeling of the
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endometrium and the tolerant milieu required for fetal acceptance.
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Aberrant phenotypes of decidual macrophages have been reported to
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associate with pathological pregnancy in human [15,16]. Decidual
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M2-like macrophages were reduced in preterm pregnancies compared
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with term pregnancies and decidual macrophages expressed high levels of
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tumor necrosis factor (TNF) and interleukin (IL)-12 during spontaneous
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preterm labor [15]. In the deciduas of patients with unexplained recurrent
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miscarriages, macrophage expression of CD80 and CD86 was higher than
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that in normal control women during early pregnancy; whereas the
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expression of IL-10 was lower [16]. In the abortion-prone (female CBA ×
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male DBA) murine mating model, the polarization patterns of placental
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macrophages were also characterized mainly by M1 phenotypes [17].
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M1-activated macrophages may damage the conceptus via production of
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nitric oxide and TNF-α [18].
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Most T. gondii strains isolated from humans and animals in North
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America and Europe have been grouped into three predominant clonal
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lineages (types I, II and III). These strains differ in virulence in mice and
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likely cause different sequelae in humans [19]. In our previous study, we
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have reported that the infection of T. gondii RH strain (type I) can
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down-regulated LILRB4 in decidual macrophages and contribute their
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functional polarization during abnormal pregnancy [20]. Nevertheless,
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the majority of human toxoplasmosis is due to infection by type II strains
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which are the most prevalent in nature [21,22], and the effect of infection
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with T. gondii type II strains on the polarization of decidual macrophages
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need further research. In the present study, an adverse pregnancy outcome
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mouse model induced by T. gondii PRU strain (Type II) infection in the
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first trimester was used to explore the immunopathology mechanism
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related to decidual macrophages.
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2. Materials and methods
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2.1. Mice and mating
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C57BL/6 mice (8–10 weeks old) were purchased from Beijing Weitong
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Lihua Experimental Animals Technical Co., Ltd. All mice were housed
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(five mice/cage) at 20°C–24°C in a specific-pathogen-free animal room
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on a 12-h light–dark cycle, with free access to food and water. Following
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overnight cohabitation with males at a ratio of 2:1, females with a vaginal
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plug, considered as gestational day (gd) 0, were segregated and
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randomized to the infection group or control group. The following animal
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experiments were approved by the Institutional Animal Experimental
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Ethics Committee of Binzhou Medical University.
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2.2. Toxoplasma gondii
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T. gondii PRU strain (Type II) was kindly provided by Professor
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Jilong Shen, Department of Pathogen Biology, Anhui Medical University,
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Anhui, China and was used to infect the mice. Cysts of T. gondii PRU
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strain were harvested from the brains of Kunming mice chronically
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infected 30–45 days earlier with approximately 20 cysts.
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2.3. Infection and pregnancy outcomes
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Pregnant mice in the infection group were perorally inoculated with
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15 cysts in 300 µl phosphate-buffered saline (PBS) on gd 4, whereas
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control mice were treated with 300 µl PBS solution. On gd 14, the mice 5
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were euthanatized by cervical dislocation, the uteri were removed, and
141
the total numbers of normal and abnormal embryos were recorded. The
142
abnormal embryos were identified by their small size, hemorrhagic and
143
necrotic appearance, or complete resorption compared with normal
144
embryos. The adverse pregnancy rate was calculated as the ratio of
145
abnormal embryo numbers to total embryo numbers.
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2.4. Histopathological and immunohistochemical analysis
Following euthanasia on gd 14, randomly selected uteri and
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placentae of pregnant mice were fixed in 4% paraformaldehyde/PBS and
150
routinely processed for paraffin embedding and sectioning. Tissue
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sections were cut at 4 µm and stained with hematoxylin and eosin (H & E;
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Boster Biotechnology, Wuhan, China) for histopathological analysis.
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Sections were examined on a photomicroscope.
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For immunohistochemistry assay, tissue sections were deparaffinized
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and rehydrated. Heat-induced antigen retrieval was performed with 0.01
156
M citrate buffer (pH 6.0; Boster Biotechnology) at 95°C for 20 min.
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Endogenous peroxidase was blocked by 3% hydrogen peroxide in
158
methanol. Sections were incubated with 2% bovine serum albumin for 30
159
min at 37°C to block non-specific binding sites and with anti-mouse
160
iNOS polyclonal antibody (mAb) (1:50 dilution; Proteintech, Wuhan,
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China) overnight at 4°C. Immunohistochemical staining was detected
162
using
163
tetrahydrochloride (both from Boster Biotechnology). The sections were
164
counterstained with Mayer’s hematoxylin and examined under a light
165
microscope.
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a
SABC
kit
and
developed
with
diaminobenzidine
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2.5. Cell preparation and flow cytometry Single-cell suspensions were prepared from placental and uterine 6
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tissues by dissecting the tissues into 1–3-mm pieces. The pieces were
170
digested with 0.15% trypsin and 25 U/ml DNase I (both from
171
Sigma-Aldrich, St. Louis, MO, USA) in RPMI 1640 (Hyclone, Logan,
172
UT, USA) at 37°C for 45 min under gentle stirring. Subsequently, the
173
samples were ground and filtered through a 75-µm pore size sieve.
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Mononuclear cells were isolated by Ficoll–Hypaque density gradient
175
centrifugation.
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Fc receptors of cells were blocked with 0.5 µg of anti-CD16/32 mAb
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(BD Pharmingen, San Diego, CA, USA) in 100 µl FACS buffer (3% FBS
178
and 0.1% sodium azide in PBS, pH 7.2) for 20 min at 4°C. The following
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fluorophore-conjugated anti-mouse mAbs were used: anti-F4/80-PE-Cy7,
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anti-CD206-PE, anti-MHC-II (I-A/I-E)-FITC (all from BioLegend, San
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Diego, CA, USA), anti-CD80-PE, and anti-CD86-PE mAbs (both from
182
BD Pharmingen). The blocked cells were incubated with mAbs at 4°C in
183
darkness for 30 min and subsequently washed twice with PBS. For
184
intracellular cytokine staining, cells were cultured with Leukocyte
185
Activation Cocktail and BD GolgiPlug (both from BD Biosciences,
186
Franklin Lakes, NJ, USA) for 4–6 h at 37°C, according to the
187
manufacturer’s instructions. After staining of F4/80, the cells were fixated
188
and permeabilized in 1× Fix/Perm buffer (Invitrogen, Carlsbad, CA, USA)
189
for 30 min. Subsequently, the cells were stained with anti-mouse
190
TNF-α-FITC (BioLegend), anti-IL-12p70-PE, and IL-10-PE (both from
191
BD Pharmingen) in darkness for 30 min at 4°C. In parallel, the
192
corresponding isotypes were used as controls. Flow cytometric data were
193
acquired using a BD FACSCantoTM II flow cytometer and analyzed using
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the FACSDiva software (BD Biosciences).
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2.6. Enzyme-linked immunosorbent assay (ELISA) Homogenates of placental tissues were prepared in PBS with a 7
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homogenizer and then centrifuged at 12,000 g for 30 min at 4°C.
199
Supernatants were collected and stored at −80°C until analysis. TNF-α,
200
IL-12p70, and IL-10 were measured using ELISA kits (Lengton
201
Bioscience,
202
instructions. The concentration of cytokines in the placental tissue
203
homogenates was calculated from a standard curve of each murine
204
recombinant cytokine. Each measurement was performed in triplicate.
China),
according
205
2.7. Magnetic cell sorting and Western blotting
the
manufacturer’s
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The mononuclear cells of mice uteri and placentae were acquired as
208
described above, and F4/80 positive cells were sorted with anti-F4/80
209
MicroBeads (Miltenyi Biotec, Bergish Gladbach, Germany), according to
210
the manufacturer’s instructions. In all cases, the purity of sorted cells was
211
at least 80%, as determined by flow cytometry.
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Protein extraction from sorted macrophages was performed in a lysis
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buffer (lysis buffer/PMSF = 16:1) (Beyotime, Shanghai, China). The cell
214
lysates were subsequently centrifuged at 12,000 g for 15 min at 4°C. The
215
denatured protein samples (20 µg protein/lane) were separated by
216
standard SDS-PAGE with 10% separating gel and transferred onto a
217
PVDF membrane (Merck Millipore, Billerica, MA, USA). After blocking
218
with 5% skim milk, the membrane was probed using purified anti-mouse
219
iNOS and arginase-1 mAbs (both 1:2,000 dilution; BD Pharmingen).
220
β-actin was stained with antibody (1:5,000 dilution; Santa Cruz, Dallas,
221
TX, USA) as control. The samples were labeled using horseradish
222
peroxidase-labeled secondary antibodies (1:10,000 dilution; Boster), and
223
chemiluminescence was evaluated using an enhanced chemiluminescence
224
kit (Roche Diagnostics, Indianapolis, IN, USA). The results were
225
analyzed using the Image J 1.46 software (NIH image, USA).
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2.8. Data analysis All data are presented as means ± standard deviation (SD). Statistical
229
analyses were performed using the SPSS 16.0 statistical software package
230
(SPSS, Inc., Chicago, IL, USA). The unpaired Student’s t-test was used to
231
evaluate differences between the two groups. P values of <0.05 or <0.01
232
were considered as significant or very significant, respectively.
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3. Results
235
3.1. Establishing an animal model with T. gondii infection-induced
236
adverse pregnancy outcome
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In the present study, pregnancy outcomes were observed on gd 14
238
after 10 days of T. gondii infection. The pregnant mice infected with T.
239
gondii PRU strain showed malaise, extrados, and erected fur, whereas the
240
control mice did not show discernible abnormal symptoms. Adverse
241
pregnancy outcomes occurred following T. gondii infection (Fig. 1A and
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1B).
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H & E staining of paraffin sections showed that infected mice had
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obvious congestion in the placenta and a larger intervillous space (Fig. 1F
245
and 1G) compared with the control mice (Fig. 1C and 1D). Furthermore,
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there were numerous inflammatory cells with round nuclei existing in the
247
placental villi of the infected pregnant mice (Fig. 1H), whereas few
248
inflammatory cells were found in the control samples (Fig. 1E).
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3.2. T. gondii infection upregulated the expression of M1 functional
251
molecules in decidual macrophages and downregulated the expression of
252
M2
253
The expression of M1 (CD80 and CD86) and M2 (CD206 and
254
MHC-II) was analyzed using flow cytometry to investigate the effect of T.
255
gondii infection on the surface functional molecules of decidual 9
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macrophages. The expression of CD80 and CD86 was upregulated,
257
whereas the expression of CD206 and MHC-II was downregulated in the
258
infection group compared with the control group (Fig. 2).
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3.3. T. gondii infection increased the expression of iNOS and decreased
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arginase-1 in decidual macrophages
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To determine the effect of T. gondii infection on L-arginine
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metabolism of decidual macrophages, the expression of iNOS and
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arginase-1 in the sorted decidual macrophages was analyzed using
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Western blotting. Compared with the control group, iNOS expression in
266
the infection group was obviously increased, whereas arginase-1
267
expression was significantly decreased (Fig. 3A and 3B).
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Immunohistochemical staining was also employed to determine the
269
expression of iNOS in the placenta and uterus. In our results, iNOS
270
expression was undetected in the control mice (Fig. 3C and 3D).
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Following T. gondii infection, iNOS-positive expression was observed in
272
the interface of uterus and placenta, in which macrophages reside (Fig.
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3E and 3F).
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3.4. T. gondii infection altered the cytokine secretion of decidual
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macrophages
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Flow cytometry was used to investigate the effect of T. gondii
278
infection on cytokine secretion of decidual macrophages. Following T.
279
gondii infection, levels of TNF-α, IL-12, and IL-10 in decidual
280
macrophages were significantly increased. However, the ratios of
281
TNF-α/IL-10 and IL-12/IL-10 in the infection group were higher than
282
those in the control group. These results suggest that T. gondii infection
283
altered the secretion pattern of cytokines related with M1/M2 polarization
284
of decidual macrophages (Fig. 4A–4C). 10
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In addition, cytokines in the supernatant of placental homogenates
286
were also measured using ELISA. Levels of TNF-α, IL-12, and IL-10 in
287
the infection group were higher than those in the control group. Moreover,
288
the ratios of TNF-α/IL-10 and IL-12/IL-10 in the infection group were
289
also higher than those in the control group. The change in cytokine
290
secretion pattern in placentae was consistent with that observed in the
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decidual macrophages (Fig. 4D and 4E).
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4. Discussion
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Maternal immune cells, including macrophages, natural killer cells,
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dendritic cells, and regulatory T cells are regulated at the maternal–fetal
296
interface to tolerate the expression of paternal antigens by the
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semi-allogenic fetus without immune rejection. Dysfunction of these
298
immune cells or dysregulation of the maternal–fetal immune tolerance
299
may lead to miscarriage, preterm delivery, or severe complications, such
300
as preeclampsia and fetal growth restriction [23]. T. gondii is one of the
301
TORCHES (toxoplasmosis, rubella, cytomegalovirus, herpes simplex,
302
and syphilis) pathogens that cause severe adverse pregnancy outcomes
303
when contacted during pregnancy [24]. Our previous studies have
304
demonstrated that T. gondii infection may cause aberrant activation of
305
natural killer cells and dendritic cells in the deciduas and lead to
306
dysfunction of regulatory T cells, which are closely associated with T.
307
gondii infection-induced adverse pregnancy outcomes [5,6,25]. In the
308
present study, we investigated the effect of T. gondii PRU strain infection
309
on decidual macrophage polarization and aimed at providing new insight
310
on the immune mechanism linked to adverse pregnancy outcomes
311
induced by this pathogen.
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Macrophages are vital immune cells in the deciduas and
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predominantly display an M2 phenotype. Decidual macrophages typically 11
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express lower levels of M1 functional molecules (CD80 and CD86) and
315
higher levels of M2 functional molecules (CD206 and MHC-II) to sustain
316
maternal–fetal immune tolerance during normal pregnancy [26,27]. On
317
the contrary, abnormal expression of these molecules has been associated
318
with the pathology of pregnancy. For example, upregulated CD80 and
319
CD86 expression in macrophages has been reported in the deciduas of
320
patients with unexplained recurrent miscarriage compared with normal
321
control women during early pregnancy [16]. Moreover, a lower
322
CD206/CD68 mRNA expression ratio in first-trimester decidual tissue
323
was found in pregnancies complicated with hypertension [28].
324
Furthermore, it has reported that the combined use of anti-CD80 and
325
anti-CD86 mAbs can induce maternal tolerance of the fetus in the
326
abortion-prone CBA/J females mated with DBA/2 males [17]. However,
327
the effect of T. gondii infection on the expression of M1- and M2-related
328
functional molecules and polarization of decidual macrophages during
329
pregnancy remains unexplored. The present study employed a mouse
330
pregnancy model with T. gondii PRU strain infection to investigate the
331
effect of infection on the expression of membrane functional molecules of
332
decidual macrophages. Our results showed that T. gondii infection
333
upregulated the expression of M1 functional molecules CD80 and CD86
334
of decidual macrophages. In contrast, the infection downregulated the
335
expression of M2 functional molecules CD206 and MHC-II. This T.
336
gondii infection-induced change in the decidual macrophage phenotype
337
enhanced the immune activity of the decidual macrophages, weakened
338
the maternal–fetal tolerance, and may have contributed to the occurrence
339
of adverse pregnancy outcomes.
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At the most fundamental level, M1/M2 polarity of macrophages is
341
driven by arginine metabolism mediated by iNOS and arginase-1 [11].
342
M1 macrophages are characterized by the expression of iNOS and the 12
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production of NO, an important effector for their microbicidal activity
344
[29]. However, a high level of iNOS expression and NO production may
345
induce immunopathology due to its autotoxic effect [30]. Excess iNOS
346
expression may suppress placental vascular development, which is
347
deleterious to the developing fetus [31]. It has reported that the
348
expression of iNOS in human placentae was not detected in normal
349
pregnancy [32]. Nevertheless, our results suggested that the expression of
350
iNOS in decidual macrophages can be induced by T. gondii infection. The
351
T. gondii-induced high expression of iNOS in decidual macrophages may
352
enhance their cytotoxic effect and consequently contribute to adverse
353
pregnancy outcomes. On the contrary, M2 macrophages do not produce
354
NO and express high levels of arginase-1, which catalyzes the production
355
of polyamines [31]. Arginase-1 antagonizes iNOS activity in arginine
356
metabolism by competing for arginine and directly inhibits iNOS [33].
357
Arginase-1 enhances the bioavailability of L-arginine and promotes
358
polyamine synthesis, which is involved in immunosuppression and
359
placental development during pregnancy [34,35]. In the present study, T.
360
gondii infection significantly decreased arginase-1 expression in decidual
361
macrophages. This result indicated that the downregulated arginase-1
362
expression by T. gondii infection may impair its antagonism against iNOS
363
activity and the maternal–fetal immune tolerance. Thus, T. gondii
364
infection skewed decidual macrophages toward M1 by inducing iNOS
365
synthesis and away from M2 decidual macrophages by downregulating
366
arginase-1 expression. Finally, the bias of M2 decidual macrophages
367
toward M1 may contribute to the occurrence of adverse pregnancy
368
outcomes caused by T. gondii infection.
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Decidual macrophages also showed an M2 polarized cytokine
370
secretion pattern with abundant production of IL-10 [36]. IL-10 is a
371
typical anti-inflammatory cytokine, playing a vital role in the maternal 13
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immune tolerance of an allogeneic fetus [37]. IL-10 deficiency leads to
373
fetal resorption and preterm birth in pregnant mice when challenged with
374
low doses of LPS or CpG [38,39]. Of note, administration of recombinant
375
IL-10 reverses or alleviates symptoms of adverse pregnancy outcomes in
376
animal models [37]. In a previous study, we showed that treatment with
377
recombinant IL-10 also alleviated adverse pregnancy outcome induced by
378
T. gondii infection in mice [40]. Decidual macrophages also produce
379
pro-inflammatory cytokines, such as TNF-α and IL-12, in normal
380
pregnancy [41]. But increase of these cytokines, especially TNF-α, is
381
associated with several obstetric disorders, such as preeclampsia, fetal
382
growth restriction, preterm labor, and spontaneous and recurrent abortion
383
[42].
384
macrophages directly induce trophoblast cell apoptosis and exacerbate
385
adverse outcomes during pregnancy [43]. In this study, levels of IL-10,
386
TNF-α, and IL-12 production in decidual macrophages were determined
387
by flow cytometry. We found that the expression levels of IL-10, IL-12,
388
and TNF-α in decidual macrophages were all increased following T.
389
gondii infection. However, TNF-α and IL-12 levels were higher
390
compared with IL-10 levels based on TNF-α/IL-10 and IL-12/IL-10 ratio
391
analysis. Furthermore, measurement of cytokines in the supernatant of
392
placental homogenates through ELISA yielded consistent findings with
393
those of cytokines secreted by decidual macrophages. Thus, T. gondii
394
infection mainly increased pro-inflammatory cytokines TNF-α and IL-12,
395
which
396
implantation sites and further damage the developing embryo. The
397
imbalance of M1 and M2 cytokine production in decidual macrophages
398
may play an important role in adverse pregnancy outcomes induced by T.
399
gondii infection.
400
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cytokines
produced
by
M1-polarized
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can
promote
cytotrophoblast
cell
apoptosis/necrosis
in
Taken together, our data indicated that T. gondii infection caused the 14
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bias of M2-like decidual macrophages toward M1 through an increase in
402
the expression levels of CD80, CD86, iNOS, TNF-α, and IL-12 and a
403
decrease in CD206, MHC-II, and arginase-1 levels. The skewing of
404
decidual macrophage polarization at the maternal–fetal interface may
405
result in weakening of the immunosuppressive microenvironment,
406
thereby contributing to adverse pregnancy outcomes induced by T. gondii
407
infection.
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The authors declare that they have no competing interests.
411 412
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Conflict of interest
Acknowledgments
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This work was supported in part by grants from the National Natural
414
Science Foundation of China (81401687, 81672049) and Taishan Scholar
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Foundation.
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Figure legends
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Fig. 1. Adverse pregnancy outcomes caused by T. gondii infection. (A)
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Representative images of mice and uteri from T. gondii-infected mice and
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controls are shown. Noticeable adverse pregnancy outcomes occurred in
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mice infected with T. gondii PRU stain compared with the control mice.
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(B) Following 10 days of infection, the adverse pregnancy rate was
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calculated as the ratio of abnormal embryo numbers to total embryo
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numbers (**P < 0.01). (C–H) H & E staining of paraffin sections from
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control and infected mice on gd 14. (C) The control mice showed normal
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aspects of the uterus (U) and placenta (P). (F) Infected mice showed
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noticeable hyperemia in the placenta. (D, G) The images also showed
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different hyperemia in the placentae of infected and control mice. (E) The
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placentae of control mice showed little inflammatory cell infiltration in
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the placental villi. (H) The placentae of infected mice showed greater
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inflammatory cell infiltration (arrows) in the placental villi.
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Fig. 2. T. gondii infection altered the expression of M1 and M2
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functional molecules in decidual macrophages. Flow cytometry was
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used to measure the expression levels of CD80 (A), CD86 (B), CD206 (C)
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and MHC-II (D), and anti-F4/80 mAb was used to gate decidual
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macrophages. Data are expressed as mean ± SD obtained from six mice
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in each group (**P < 0.01).
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Fig. 3. T. gondii infection affected the expression of iNOS and
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arginase-1 in decidual macrophages. (A) The expression of iNOS and
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arginase-1 in decidual macrophages was analyzed by Western blotting. (B)
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The relative expression of iNOS and arginase-1 is shown. Data are
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represented as mean ± SD of three independent experiments (*P < 0.05,
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**P < 0.01). (C–F) The expression of iNOS in the placenta (P) and uterus 21
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(U) was also analyzed by immunohistochemical staining. iNOS-positive
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cells in tissues were identified by brown staining. Noticeable iNOS
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expression was observed at the interface between uterus and placenta in
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the T. gondii-infected mice (E, F), whereas iNOS expression was
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undetectable in the control mice (C, D). The figure is representative of
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five independent experiments.
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Fig. 4. The secretion pattern of cytokines in decidual macrophages
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was altered following T. gondii infection. (A) The expression of
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cytokine TNF-α, IL-12, and IL-10 was assessed by flow cytometry. Cells
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are gated on F4/80-positive cells, and the scatter diagrams of cytokines
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are shown. (B) The positive rates of TNF-α, IL-12, and IL-10 expressed
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in decidual macrophages were counted. Data are presented as mean ± SD
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obtained from six mice in each group. (C) The ratios of TNF-α/IL-10 and
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IL-12/IL-10 in decidual macrophages are shown. (D) Levels of TNF-α,
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IL-12, and IL-10 in supernatants of placental homogenates were
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determined using ELISA. Each measurement was performed in triplicate.
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Data are represented as mean ± SD obtained from six mice in each group.
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(E) The ratios of TNF-α/IL-10 and IL-12/IL-10 in supernatants of
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placental homogenates are also shown. *P < 0.05, **P < 0.01.
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Highlights
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T. gondii infection changes functional molecule expression in decidual macrophages. T. gondii infection affects iNOS and arginase-1 expression in decidual macrophages. T. gondii infection alters the pattern of cytokine secretion in decidual macrophages.
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T. gondii infection causes M1 polarization of decidual macrophages.
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The bias of M1/M2 polarization contributes to adverse pregnancy outcomes.