Hormonal changes during pregnancy instigate numerous physiological changes aimed at the growth and delivery of a healthy baby. A careful balance between immunological tolerance against fetal antigens and immunity against infectious agents needs to be maintained. A three-way interaction between pregnancy hormones, the immune system and our microbiota is now emerging. Recent evidence suggests that microbial alterations seen during pregnancy may help maintain homeostasis and aid the required physiological changes occurring in pregnancy. However, these same immunological and microbial alterations may also make women more vulnerable during pregnancy and the post-partum period, especially regarding immunological and infectious diseases. Thus, a further understanding of the host-microbial interactions taking place during pregnancy may improve identification of populations at risk for adverse pregnancy outcomes.

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The immune system and microbiome in pregnancy

Bovine neosporosis, one of the main causes of reproductive failure in cattle worldwide, poses a challenge for the immune system of pregnant cows. Changes in the Th-1/Th-2 balance in the placenta during gestation have been associated with abortion. Cotyledon and caruncle cell layers form the maternal-foetal interface in the placenta and are able to recognize and induce immune responses against Neospora caninum among other pathogens. The objective of the present work was to elucidate the immunomodulation produced by high- (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates of N. caninum in bovine trophoblast (F3) and caruncular cells (BCEC-1) at early and late points after infection. Variations in the mRNA expression levels of toll-like receptor-2 (TLR-2), Th1 and Th2 cytokines (IL-4, IL-10, IL-8, IL-6, IL-12p40, IL-17, IFN-γ, TGF-β1, TNF-α), and endothelial adhesion molecules (ICAM-1 and VCAM-1) were investigated by RT-qPCR, and protein variations in culture supernatants were investigated by ELISA. A similar pattern of modulation was found in both cell lines. The most upregulated cytokines in infected cells were pro-inflammatory TNF-α (P < 0.05–0.0001) and IL-8 (P < 0.05–0.001) whereas regulatory IL-6 (P < 0.05–0.001) and TGF-β1 (P < 0.05–0.001) were downregulated in both cell lines. The measurement of secreted IL-6, IL-8 and TNF-α confirmed the mRNA expression level results. Differences between isolates were found in the mRNA expression levels of TLR-2 (P < 0.05) in both cell lines and in the mRNA expression levels (P < 0.05) and protein secretion of TNF-α (P < 0.05), which were higher in the trophoblast cell line (F3) infected with the low-virulence isolate Nc-Spain1H. Neospora caninum infection is shown to favor a pro-inflammatory response in placental target cells in vitro. In addition, significant immunomodulation differences were observed between high- and low-virulence isolates, which would partially explain the differences in virulence.

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Immune response profile of caruncular and trophoblast cell lines infected by high- (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates of Neospora caninum

Maternal natural killer cells at the intersection between reproduction and mucosal immunity.

Autophagy is a complex degradative process by which eukaryotic cells capture cytoplasmic components for subsequent degradation through lysosomal hydrolases. Although this catabolic process can be triggered by a great variety of stimuli, action in cells varies according to cellular context. Autophagy has been previously linked to disease development modulation, including cancer. Autophagy helps suppress cancer cell advancement in tumor transformation early stages, while promoting proliferation and metastasis in advanced settings. Oncoviruses are a particular type of virus that directly contribute to cell transformation and tumor development. Extensive molecular studies have revealed complex ways in which autophagy can suppress or improve oncovirus fitness while still regulating viral replication and determining host cell fate. This review includes recent advances in autophagic cellular function and emphasizes its antagonistic role in cancer cells.

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Autophagy in Viral Development and Progression of Cancer.

Since murine cytomegalovirus (MCMV) was first described in 1954, it has been used to model human cytomegalovirus (HCMV) diseases. MCMV is a natural pathogen of mice that is present in wild mice populations and has been associated with diseases such as myocarditis. The species-specific nature of HCMV restricts most research to cell culture-based studies or to the investigation of non-invasive clinical samples, which may not be ideal for the study of disseminated disease. Initial MCMV research used a salivary gland-propagated virus administered via different routes of inoculation into a variety of mouse strains. This revealed that the genetic background of the laboratory mice affected the severity of disease and altered the extent of subsequent pathology. The advent of genetically modified mice and viruses has allowed new aspects of disease to be modeled and the opportunistic nature of HCMV infection to be confirmed. This review describes the different ways that MCMV has been used to model HCMV diseases and explores the continuing difficulty faced by researchers attempting to model HCMV congenital cytomegalovirus disease using the mouse model.

https://www.mdpi.com/1422-0067/22/1/214/pdf

A Review of Murine Cytomegalovirus as a Model for Human Cytomegalovirus Disease-Do Mice Lie?

Increasing evidence has revealed that maternal cytomegalovirus (CMV) infection may be associated with neurodevelopmental disorders in offspring. Potential relevance between the placental inflammation and CMV-related autism has been reported by clinical observation. Meanwhile, abnormal expression of Toll-like receptor 2 (TLR2) and TLR4 in placenta of patients with chorioamnionitis was observed in multiple studies. IL-6 and IL-10 are two important maternal inflammatory mediators involved in neurodevelopmental disorders. To investigate whether murine CMV (MCMV) infection causes alterations in placental IL-6/10 and TLR2/4 levels, we analyzed the dynamic changes in gene expression of TLR2/4 and IL-6/10 in placentas following acute MCMV infection. Mouse model of acute MCMV infection during pregnancy was created, and pre-pregnant MCMV infected, lipopolysaccharide (LPS)-treated and uninfected mice were used as controls. At E13.5, E14.5 and E18.5, placentas and fetal brains were harvested and mRNA expression levels of placental TLR2/4 and IL-6/10 were analyzed. The results showed that after acute MCMV infection, the expression levels of placental TLR2/4 and IL-6 were elevated at E13.5, accompanied by obvious placental inflammation and reduction of placenta and fetal brain weights. However, LPS 50 μg/kg could decrease the EL-6 expression at E13.5 and E14.5. This suggests that acute MCMV infection during pregnancy could up-regulate the gene expression of TLR2/4 in placental trophoblasts and activate them to produce more proinflammatory cytokine IL-6. High dose of LPS stimulation (50 μg/kg) during pregnancy can lead to down-regulation of IL-6 levels in the late stage. Imbalance of IL-6 expression in placenta might be associated with the neurodevelopmental disorders in progeny.

Maternal Murine Cytomegalovirus Infection during Pregnancy Up-regulates the Gene Expression of Toll-like Receptor 2 and 4 in Placenta.

Background Cytomegalovirus (CMV) induced autophagy affects virus replication and survival of the infected cells. The purpose of this study was to investigate the role of autophagy inhibition by 3-methyladenine (3-MA) on murine cytomegalovirus (MCMV) replication and whether it is associated with caspase-3 dependent apoptosis. Methods The eyecup isolated from adult C57BL/6J mice (6-8 weeks old) and mouse embryo fibroblast cells (MEFs) were infected with MCMV K181 strain, followed by the treatment of 3-methyladenine (3-MA), chloroquine(CQ) or rapamycin to block or stimulate autophagy. Results In cultured MEFs, the ratio of LC3I/II was reduced at 24 hours post infection (h.p.i.), but was increased at 48h.p.i. In the eyecup culture, LC3I/II ratio was also decreased at 4 and 7days post infection (d.p.i.). In addition, caspase-3 cleavage was increased at 48h.p.i. in MEFs and also elevated in MCMV infected eyecups at 4, 7, 10 and 14d.p.i. 3-MA treatment significantly inhibited the virus replication in MEFs and eyecups. The expression of early antigen (EA) of MCMV was also decreased in MEFs and eyecups. Meanwhile, cleaved caspase-3 dependent cell death was promoted with the presence of 3-MA in MCMV infected MEFs and eyecups, while RIPK1/RIPK3/MLKL pathway was inhibited by 3-MA in eyecups. Conclusions Inhibition of autophagy by 3-MA restricts virus replication and promotes caspase-3 dependent apoptosis in the eyecup and MEFs with MCMV infection. It can be explained that during the early period of MCMV infection, suppressed autophagy process directly reduced virus release, but later caspase-3 dependent apoptosis dominated and resulted in decreased virus replication. This article is protected by copyright. All rights reserved.

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Inhibition of autophagy by 3‐methyladenine restricts murine cytomegalovirus replication

The purpose of this study was to determine whether autophagy regulates the expression of human cytomegalovirus (HCMV) immediately early two viral protein (IE2). Rapamycin and 3-methyladenine (3-MA) were used to stimulate or suppress autophagy during HCMV infection. UL122 recombinant plasmid was transfected to overexpress IE2 and small interference RNA against autophagy-related protein 3 (ATG3) was used to knockdown ATG3. Western blot was performed to measure the expression of viral proteins and autophagy levels. Immunofluorescence was used to detect the immediately early 1 viral protein (IE1) expression. In human embryonic lung fibroblasts, infection of HCMV promotes the lipidation of light chain 3 (LC3) at 6 and 24 hours post infection (hpi), which was accompanied by the increased expression of viral protein IE2. When only IE2 was overexpressed via UL122 recombinant plasmid transfection without HCMV infection, the autophagy hallmarks LC3II and ATG3 were upregulated. Furthermore, viral protein IE2 expression was reduced at 24 and 48 hpi either by the treatment of autophagy inducer rapamycin or by the inhibitor 3-MA before HCMV infection. At the same time, small interference ATG3 transient transfection, used to suppress autophagy, significantly inhibited IE2 expression. However, when 3-MA was used to regulate autophagy levels after HCMV infection, expression of IE2 and IE1 were both decreased, while autophagy inducer rapamycin treatment after HCMV infection increased IE2 expression slightly. IE2 was involved in autophagy induced by HCMV infection and blocking autophagy could inhibit the expression of HCMV viral protein IE2, which might be one way for autophagy to restrict HCMV replication.

The role of autophagy in human cytomegalovirus IE2 expression

Absent in melanoma 2 (AIM2) inflammasome is a crucial link bridging the innate host defense and the subsequent adaptive immunity when activated by exogenous double stranded DNA (dsDNA). Through establishing models of disseminated murine cytomegalovirus (MCMV) infection in BALB/c and C57BL/6 mice, we evaluated dynamic expression of AIM2 inflammasome components and its relationship with pathological damage and viral replication, trying to figure out whether AIM2 inflammasome is related to the chronic mechanism of MCMV. BALB/c and C57BL/6 mice were sacrificed on day 0, 1, 3, 7, 14 and 28 post infection. Expression levels of AIM2, pro-caspase-1, caspase-1 p20, pro-IL1β and mature IL1β in primary peritoneal macrophages (PMs) and spleens were detected by Western blotting. Contents of IL18 in the serum were detected by ELISA. Pathological examinations of livers were performed, and mRNA levels of MCMV glycoprotein B (gB) in salivary glands also assessed. Results showed that expression levels of AIM2 in PMs and spleens of C57BL/6 mice increased on day 3, even continued to day 28; caspase-1 p20 and mature IL1β increased on day 7, 14 and 28; the persistently high expression of IL18 in the serum started on day 1, showing a double peak curve. As for BALB/c mice, expression of AIM2 in PMs increased on day 1 and day 7, while contents of AIM2 in spleens increased on day 1 and day 3; caspase-1 p20 and mature IL1β merely increased 7 days fter infection. Thereafter, expression levels of AIM2, caspase-1 p20, mature IL1β and IL18 were limited; the duration of AIM2 inflammasome activation in BALB/c mice was much shorter than that in C57BL/6 mice. The severer pathological damage and more viral replications in BALB/c mice further proved the deficient antiviral immunity to MCMV. In conclusion, the activation of AIM2 inflammasome in BALB/c mice was short-lived, which is quite possibly related to the chronicity of MCMV infection.

Short-lived AIM2 Inflammasome Activation Relates to Chronic MCMV Infection in BALB/c Mice.

Autophagy is involved in the pathogenesis of multiple pathogen infection Previous studies have reported that human cytomegalovirus (CMV) activates autophagy in the early stage of infection and then inhibits autophagy Little is known about the role of autophagy in murine CMV (MCMV) infection, especially in MCMV-induced hepatitis The purpose of this study is to investigate the role of autophagy in MCMV hepatitis BALB/c mice were infected with MCMV and a series of experiments involving western blot, immunofluorescence, immunohistochemistry, HE whereas decreased transcription level of MCMV gB, decreased expression levels of IL-1β, AST and ALT, increased expression level of type I IFN-α, as well as alleviated liver pathological injury were detected in chloroquine (CQ)-treated infected group on day 3 pi In conclusion, autophagy is inhibited through activating the PI3K/Akt/mTOR pathway in the liver of BALB/c mice during MCMV infection, and autophagy may promote MCMV replication and aggravate liver pathological damage and inflammation Further understanding of the interactions between autophagy and MCMV infection and its potential mechanism may bring new important cues to the control of MCMV infection and antiviral therapy

Lin-Lin Zhang

Papers

Maternal Murine Cytomegalovirus Infection during Pregnancy Up-regulates the Gene Expression of Toll-like Receptor 2 and 4 in Placenta.

Inhibition of autophagy by 3‐methyladenine restricts murine cytomegalovirus replication

The role of autophagy in human cytomegalovirus IE2 expression

Short-lived AIM2 Inflammasome Activation Relates to Chronic MCMV Infection in BALB/c Mice.

The Role of Autophagy in Murine Cytomegalovirus Hepatitis.