Mechanisms of immune regulation by the placenta: Role of type I interferon and interferon-stimulated genes signaling during pregnancy.

Pregnancy is a unique condition where the maternal immune system is continuously adapting in response to the stages of fetal development and signals from the environment. The placenta is a key mediator of the fetal/maternal interaction by providing signals that regulate the function of the maternal immune system as well as provides protective mechanisms to prevent the exposure of the fetus to dangerous signals. Bacterial and/or viral infection during pregnancy induce a unique immunological response by the placenta, and type I interferon is one of the crucial signaling pathways in the trophoblast cells. Basal expression of type I interferon-ß and downstream ISGs harbors physiological functions to maintain the homeostasis of pregnancy, more importantly, provides the placenta with the adequate awareness to respond to infections. The disruption of type I interferon signaling in the placenta will lead to pregnancy complications and can compromise fetal development. In this review, we focus the important role of placenta-derived type I interferon and its downstream ISGs in the regulation of maternal immune homeostasis and protection against viral infection. These studies are helping us to better understand placental immunological functions and provide a new perspective for developing better approaches to protect mother and fetus during infections.

Next generation of immune checkpoint molecules in maternal-fetal immunity.

Successful pregnancy is a unique situation requires the maternal immune system to recognize and tolerate a semi-identical fetus and allow normal invasion of trophoblast cells. Although efforts have been made, the deep mechanisms of the maternal-fetal crosstalk have not yet been fully deciphered. Immune checkpoint molecules (ICMs) are a group of negative modulators of the immune response that avoid immune damage. They have been extensively studied in the fields of oncology and transplantation, while the latest evidence suggests that they are closely associated with pregnancy outcomes via multiple inhibitory mechanisms. Although studies have mostly demonstrated the regulatory role of the well-known PD-1, CTLA-4 at the maternal-fetal interface, what is unique about the newly discovered multiple ICMs remains a mystery. Here, we review the latest knowledge on ICMs, focusing on the first generation of checkpoints (PD-1, CTLA-4) and the next generation (Tim-3, Tigit, Lag-3, VISTA) highlighting their immunoregulatory roles in maternal-fetal tolerance and decidual vascular remodeling, and their involvement in pathological pregnancies. The content covers three aspects: the characteristics they possess, the dynamic expression profile of their expression at the maternal-fetal interface, and their involvement in pathological pregnancy. In immunotherapy strategies for pregnancy complications, upregulation of immune checkpoints may play a role. Meanwhile, the impact on pregnancy outcomes when using ICMs in clinical cancer treatment during pregnancy is a topic worth exploring. These may serve as a guide for future basic research and clinical applications of maternal-fetal immunity.

Twist1-IRF9 Interaction Is Necessary for IFN-Stimulated Gene Anti-Zika Viral Infection.

An efficient immune defense against pathogens requires sufficient basal sensing mechanisms that can deliver prompt responses. Type I IFNs are protective against acute viral infections and respond to viral and bacterial infections, but their efficacy depends on constitutive basal activity that promotes the expression of downstream genes known as IFN-stimulated genes (ISGs). Type I IFNs and ISGs are constitutively produced at low quantities and yet exert profound effects essential for numerous physiological processes beyond antiviral and antimicrobial defense, including immunomodulation, cell cycle regulation, cell survival, and cell differentiation. Although the canonical response pathway for type I IFNs has been extensively characterized, less is known regarding the transcriptional regulation of constitutive ISG expression. Zika virus (ZIKV) infection is a major risk for human pregnancy complications and fetal development and depends on an appropriate IFN-ß response. However, it is poorly understood how ZIKV, despite an IFN-ß response, causes miscarriages. We have uncovered a mechanism for this function specifically in the context of the early antiviral response. Our results demonstrate that IFN regulatory factor (IRF9) is critical in the early response to ZIKV infection in human trophoblast. This function is contingent on IRF9 binding to Twist1. In this signaling cascade, Twist1 was not only a required partner that promotes IRF9 binding to the IFN-stimulated response element but also an upstream regulator that controls basal levels of IRF9. The absence of Twist1 renders human trophoblast cells susceptible to ZIKV infection.

Interleukin-10: a novel metabolic inducer of macrophage differentiation and subsequently contributing to improved pregnancy outcomes of mice by orchestrating oxidative phosphorylation metabolism†.

Metabolism regulates the phenotype and function of macrophages. After recruitment to local tissues, monocytes are influenced by the local microenvironment and differentiate into various macrophages depending on different metabolic pathways. However, the metabolic mechanisms underlying decidual macrophage differentiation remain unknown. Interleukin-10 (IL-10) is an important decidual macrophage inducer and promotes oxidative phosphorylation (OXPHOS) of bone marrow-derived macrophages. In this study, we mainly investigate the metabolic changes involved in IL-10-generated macrophages from monocytes using in vitro models. We demonstrate that exposure of monocytes (either peripheral or THP-1) to IL-10 altered the phenotype and function of resultant macrophages that are linked with OXPHOS changes. Interleukin-10 enhanced the mitochondrial complex I and III activity of THP-1 cell-differentiated macrophages and increased the mitochondrial membrane potential, intracellular adenosine triphosphate, and reactive oxygen species levels. Oxidative phosphorylation blockage with oligomycin changed the cell morphology of IL-10-generated macrophages and the expression levels of cytokines, such as transforming growth factor beta, tumor necrosis factor-alpha, interferon gamma, and IL-10, apart from changes in the expression level of the surface markers CD206, CD209, and CD163. Moreover, in vivo IL-10 administration reduced the lipopolysaccharide (LPS)-induced embryo resorption rate, and this effect was diminished when OXPHOS was inhibited, demonstrating that OXPHOS is important for the improved pregnancy outcomes of IL-10 in LPS-induced abortion-prone mice. Our findings provide deep insights into the roles of IL-10 in macrophage biology and pregnancy maintenance. Nevertheless, the direct evidence that OXPHOS is involved in decidual macrophage differentiation needs further investigations.

A multi-organ analysis of the role of mTOR in fetal alcohol spectrum disorders.

Alcohol exposure during gestation can lead to fetal alcohol spectrum disorders (FASD), an array of cognitive and physical developmental impairments. Over the past two and a half decades, Mammalian Target of Rapamycin (mTOR) has emerged at the nexus of many fields of study, and has recently been implicated in FASD etiology. mTOR plays an integral role in modulating anabolic and catabolic activities, including protein synthesis and autophagy. These processes are vital for proper development and can have long lasting effects following alcohol exposure, such as impaired hippocampal and synapse formation, reduced brain size, as well as cognitive, behavioral, and memory impairments. We highlight recent advances in the field of FASD, primarily with regard to animal model discoveries and discuss the interaction between alcohol and mTOR in the context of various tissues, including brain, placenta, bone, and muscle, with respect to developmental alcohol exposure paradigms. The current review focuses on novel FASD research within the context of the mTOR signaling and sheds light on mechanistic etiologies at various biological levels including molecular, cellular, and functional, across multiple stages of development and illuminates the dichotomy between the different mTOR complexes and their unique signaling roles.

A newly intervention strategy in preeclampsia: Targeting PD-1/Tim-3 signaling pathways to modulate the polarization of decidual macrophages.

Programmed cell death-1 (PD-1) and T-cell immunoglobulin mucin-3 (Tim-3) are important immune checkpoint receptors that prevent an overreacted maternal immune response to fetal antigens during pregnancy. Disruption of complex immune regulation mechanisms is associated with adverse pregnancy outcomes, including preeclampsia (PE). Our recent study showed that the Tim-3 pathway was involved in the regulation of decidual macrophage polarization. Decidual macrophages polarized to the M1 phenotype may impair uterine vessel remodeling during placentation, accounting for the occurrence of PE. Co-blockade of the PD-1/Tim-3 pathway was shown to successfully control tumor growth in preclinical cancer models. However, the effects of activating both PD-1 and Tim-3 pathways as a combined intervention strategy in PE are never reported. Herein, we observed the skew of decidual macrophage polarization toward the M1 phenotype in patients with PE and lipopolysaccharide (LPS)-induced PE-like rat model. Moreover, we found that the activation of PD-1/Tim-3 pathway by using PD-L1 and Gal-9 fusion proteins could alleviate the manifestation of the LPS-induced PE-like rats and protect their offspring. Compared with the single intervention, the combination of PD-L1and Gal-9 fusion proteins exhibited obvious advantages in the relief of PE-like symptoms, trophoblast invasion, and fetal vascular development, indicating a synergistic effect of the activated PD-1/Tim-3 pathway. The in vitro study also revealed that the combined intervention using PD-L1 and Gal-9 fusion proteins inhibited the LPS-induced M1 macrophage polarization via the synergic activation of the ERK/GSK3ß/ß-catenin signaling pathway. Together, our findings provide the first evidence that simultaneous activation of PD-1/Tim-3 signaling pathways may have an optimal protective effect and serve as a new potential target for PE intervention.

Placenta-derived interferon-stimulated gene 20 controls ZIKA virus infection.

Zika virus is a positive-sense single-stranded RNA virus, which can be transmitted across the placenta and has adverse effects on fetal development during pregnancy. The severity of these complications highlights the importance of prevention and treatment. However, no vaccines or drugs are currently available. In this study, we characterize the IFNß-mediated anti-viral response in trophoblast cells in order to identify critical components that are necessary for the successful control of viral replication and determine whether components of the IFN-induced response can be used as a replacement therapy for ZIKA virus infection during pregnancy. We identify and characterize interferon-stimulated gene 20 (ISG20) as playing a central role in controlling Zika virus infection in trophoblast cells and successfully establish a recombinant ISG20-Fc protein that effectively decreases viral titers in vitro and in vivo by maintaining its exonuclease activity and displaying potential immune modulatory functions. Recombinant ISG20-Fc has thus the potential to be further developed as an anti-viral treatment against ZIKA viral infection in high-risk populations, particularly in pregnant women.

COVID-19: disease pathways and gene expression changes predict methylprednisolone can improve outcome in severe cases.

MOTIVATION: COVID-19 has several distinct clinical phases: a viral replication phase, an inflammatory phase and in some patients, a hyper-inflammatory phase. High mortality is associated with patients developing cytokine storm syndrome. Treatment of hyper-inflammation in these patients using existing approved therapies with proven safety profiles could address the immediate need to reduce mortality. RESULTS: We analyzed the changes in the gene expression, pathways and putative mechanisms induced by SARS-CoV2 in NHBE, and A549 cells, as well as COVID-19 lung versus their respective controls. We used these changes to identify FDA approved drugs that could be repurposed to help COVID-19 patients with severe symptoms related to hyper-inflammation. We identified methylprednisolone (MP) as a potential leading therapy. The results were then confirmed in five independent validation datasets including Vero E6 cells, lung and intestinal organoids, as well as additional patient lung sample versus their respective controls. Finally, the efficacy of MP was validated in an independent clinical study. Thirty-day all-cause mortality occurred at a significantly lower rate in the MP-treated group compared to control group (29.6% versus 16.6%, P = 0.027). Clinical results confirmed the in silico prediction that MP could improve outcomes in severe cases of COVID-19. A low number needed to treat (NNT = 5) suggests MP may be more efficacious than dexamethasone or hydrocortisone. AVAILABILITY AND IMPLEMENTATION: iPathwayGuide is available at https://advaitabio.com/ipathwayguide/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Exposure to a mixture of per-and polyfluoroalkyl substances modulates pulmonary expression of ACE2 and circulating hormones and cytokines.

Genetic and environmental factors impact on the interindividual variability of susceptibility to communicable and non-communicable diseases. A class of ubiquitous chemicals, Per- and polyfluoroalkyl substances (PFAS) have been linked in epidemiological studies to immunosuppression and increased susceptibility to viral infections, but possible mechanisms are not well elucidated. To begin to gain insight into the role of PFAS in susceptibility to one such viral infection, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), male and female C57BL/6 J mice were exposed to control water or a mixture of 5 PFAS (PFOS, PFOA, PFNA, PFHxS, Genx) for 12 weeks and lungs were isolated for examination of expression of SARS-CoV-2-related receptors Angiotensin-Converting Enzyme 2 (ACE2) and others. Secondary analyses included circulating hormones and cytokines which have been shown to directly or indirectly impact on ACE2 expression and severity of viral infections. Changes in mRNA and protein expression were analyzed by RT-qPCR and western blotting and circulating hormones and cytokines were determined by ELISA and MESO QuickPlex. The PFAS mixture decreased Ace2 mRNA 2.5-fold in male mice (p < 0.0001), with no significant change observed in females. In addition, TMPRSS2, ANPEP, ENPEP and DPP4 (other genes implicated in COVID-19 infection) were modulated due to PFAS. Plasma testosterone, but not estrogen were strikingly decreased due to PFAS which corresponded to PFAS-mediated repression of 4 representative pulmonary AR target genes; hemoglobin, beta adult major chain (Hbb-b1), Ferrochelatase (Fech), Collagen Type XIV Alpha 1 Chain (Col14a1), 5'-Aminolevulinate Synthase 2 (Alas2). Finally, PFAS modulated circulating pro and anti-inflammatory mediators including IFN-γ (downregulated 3.0-fold in females; p = 0.0301, 2.1-fold in males; p = 0.0418) and IL-6 (upregulated 5.6-fold in males; p = 0.030, no change in females). In conclusion, our data indicate long term exposure to a PFAS mixture impacts mechanisms related to expression of ACE2 in the lung. This work provides a mechanistic rationale for important future studies of PFAS exposure and subsequent viral infection.

Lipopolysaccharide-Stimulated Human Fetal Membranes Induce Neutrophil Activation and Release of Vital Neutrophil Extracellular Traps.

Preterm birth is a major contributor to neonatal mortality and morbidity, and infection is a major risk factor. Chorioamnionitis, inflammation of the placenta, and fetal membranes (FMs) are commonly observed in preterm birth and are characterized by neutrophil infiltration. However, interactions between FMs and neutrophils remain incompletely understood. The objectives of this study were to determine how FMs, with or without bacterial LPS stimulation, affect neutrophil recruitment, activation, and the formation of neutrophil extracellular traps (NETs) and to elucidate the signaling mechanisms involved. Using a combination of in vitro, ex vivo, and in vivo approaches, we show that human resting FMs can directly recruit neutrophils and induce them to produce proinflammatory factors. Furthermore, neutrophils release vital NETs in response to FM-derived factors. LPS-stimulated FMs further augmented neutrophil recruitment, inflammatory cytokine/chemokine secretion, and vital NET release and also induced reactive oxygen species production and degranulation. We demonstrate a role for FM-derived TNF-α in mediating these effects through activation of neutrophil p38 MAPK. We propose that, during infection, neutrophil recruitment and activation may neutralize pathogens, vital NET formation, and prolonged neutrophil viability, and in combination with degranulation, reactive oxygen species production and inflammatory chemokine/cytokine production may contribute to tissue injury at the maternal/fetal interface.

Epigenetic modifications working in the decidualization and endometrial receptivity.

Decidualization is a critical event for the blastocyst implantation, placental development and fetal growth and the normal term. In mice, the embryo implantation to the uterine epithelial would trigger the endometrial stromal cells to differentiate into decidual stromal cells. However, decidualization in women takes place from the secretory phase of each menstrual cycle and continues to early pregnancy if there is conceptus. Deficient decidualization is often associated with pregnancy specific complications and reproductive disorders. Dramatic changes occur in the gene expression profiles during decidualization, which is coordinately regulated by steroid hormones, growth factors, and molecular and epigenetic mechanisms. Recently, emerging evidences showed that epigenetic modifications, mainly including DNA methylation, histone modification, and non-coding RNAs, play an important role in the decidualization process via affecting the target genes' expression. In this review, we will focus on the epigenetic modifications in decidualization and open novel avenues to predict and treat the pregnancy complications caused by abnormal decidualization.

Protein kinase Cα-mediated phosphorylation of Twist1 at Ser-144 prevents Twist1 ubiquitination and stabilizes it.

Twist1 is a basic helix-loop-helix transcription factor that plays a key role in embryonic development, and its expression is down-regulated in adult cells. However, Twist1 is highly expressed during cancer development, conferring a proliferative, migratory, and invasive phenotype to malignant cells. Twist1 expression can be regulated post-translationally by phosphorylation or ubiquitination events. We report in this study a previously unknown and relevant Twist1 phosphorylation site that controls its stability. To identify candidate phosphorylation sites in Twist1, we first conducted an in silico analysis of the Twist1 protein, which yielded several potential sites. Because most of these sites were predicted to be phosphorylated by protein kinase C (PKC), we overexpressed PKCα in several cell lines and found that it phosphorylates Twist1 on Ser-144. Using a combination of immunoblotting, immunoprecipitation, protein overexpression, and CRISPR/Cas9-mediated PKCα knockout experiments, we observed that PKCα-mediated Twist1 phosphorylation at Ser-144 inhibits Twist1 ubiquitination and consequently stabilizes it. These results provide evidence for a direct association between PKCα and Twist1 and yield critical insights into the PKCα/Twist1 signaling axis that governs cancer aggressiveness.

Newly characterized decidual Tim-3+ Treg cells are abundant during early pregnancy and driven by IL-27 coordinately with Gal-9 from trophoblasts.

STUDY QUESTION: What is the mechanism of Tim-3+ regulatory T (Treg)-cell accumulation in the decidua during early pregnancy and is its disruption associated with recurrent pregnancy loss (RPL)? SUMMARY ANSWER: IL-27 and Gal-9 secreted by trophoblasts activate the Tim-3 signaling pathway in CD4+ T cells and Treg cells and so promote accumulation of Tim-3+ Treg cells, the abnormal expression of IL-27 and Gal-9 is associated with impaired immunologic tolerance in RPL patients. WHAT IS KNOWN ALREADY: Tim-3+ Treg cells are better suppressors of Teff cell proliferation, and display higher proliferative activity than Tim-3- Treg cells. Tim-3+ Treg cells are tissue-specific promoters of T-cell dysfunction in many tumors. These cells express a unique factor that influences and shapes the tumor microenvironment. STUDY DESIGN, SIZE, DURATION: The animal study included 80 normal pregnant mice. In human study, decidua tissues in the first trimester for flow cytometry analysis were collected from 32 normal pregnant women and 23 RPL patients. Placenta tissues for immunohistochemistry analysis were collected from 15 normal pregnant women. Placenta tissues for western blot analysis were collected from 5 normal pregnant women, 5 RPL patients and 5 women who have experienced one miscarriage. Blood samples for in vitro experiments were collected from 30 normal pregnant women. This study was performed between January 2017 and March 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: In this study, we investigated the kinetics of Tim-3+ CD4+ T-cell accumulation, and the proportions of Tim-3+ Treg cells throughout murine pregnancies using flow cytometry. We compared Tim-3 expression on decidual CD4+ T cells and Treg cells during normal pregnancies with expression on the same cell populations in women suffering from RPL. IL-27 and Gal-9 transcription and protein expression in the placenta were determined by RT-PCR and western blot, respectively. An in vitro co-culture model consisting of peripheral CD4+ T cells and primary trophoblasts from early pregnancy was used to mimic the maternal-fetal environment. MAIN RESULTS AND THE ROLE OF CHANCE: The percentage of Tim-3+ Treg cells present in mouse uteri fluctuates as gestation proceeds but does not change in the spleen. Levels of Tim3+ Treg cells in uteri peaked at pregnancy Day 6.5 (E 6.5), then progressively diminished, and fell to non-pregnant levels by E18.5. In pregnant mice, Tim-3+ Treg cells constituted 40-70% of Treg cells in uteri but were present at much lower abundance in spleens. About 60% of decidual Treg cells were Tim-3 positive at E6.5. Of these decidual Tim3+ Treg cells, nearly 90% were PD-1 positive. However, only about 16% of Tim3- Treg cells expressed PD-1. Blocking the Tim-3 signaling pathway decreased the proportion of Treg cells and led to embryo resorption. Moreover, much lower Tim-3 expression was observed on CD4+ T cells and Treg cells in women who had suffered from RPL at 6-9 gestational weeks compared with those who had normal pregnancies at matched gestations. In a normal pregnancy, Tim-3 expression on decidual CD4+ T cells is induced initially by IL-27. Then Gal-9-Tim-3 interaction promotes differentiation of decidual Tim-3+ CD4+ T cells into Treg cells. IL-27 and Gal-9 cooperatively induced Tim-3+ Treg cells in vitro. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We did not investigate the kinetics of human decidual Tim-3+ CD4+ T and Tim-3+ Treg cell populations throughout pregnancy due to limited availability of second and third trimester decidua. In addition, functional suppressive data on the decidual Tim-3+ Treg cells are lacking due to limited and low quantities of these cells in decidua. WIDER IMPLICATIONS OF THE FINDINGS: These findings might have therapeutic clinical implications in RPL. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by research grants from the National Natural Science Foundation of China (No. 81871186) and National Key Research & Developmental Program of China (2018YFC1003900, 2018YFC1003904). The authors declare no conflict of interest.

Covid-19 pandemic and pregnancy.

At the end of 2019, a new coronavirus disease, COVID-19, emerged and quickly spread around the world. Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), the causative virus of this disease, belongs to the ß-coronavirus family, together with SARS and middle east respiratory syndrome, and has similar biological characteristics to these viruses. For obstetricians, the susceptibility and prognoses of pregnant women and the effects of the infection on the fetus have been the focus of attention; however, at present, the seriousness of the disease in pregnant women is not apparent, and COVID-19 does not increase the rate of miscarriage, stillbirth, preterm labor or teratogenicity. Even so, carriers might transmit SARS-CoV-2 to pregnant women. Thus, we must keep in mind that all medical personnel must understand and maintain standard precautions in their clinical and laboratory practices.

The role of the PD-1/PD-L1 axis in macrophage differentiation and function during pregnancy.

STUDY QUESTION: What is the role of the programmed cell death-1 (PD-1)/PD-1 ligand-1 (PD-L1) axis in macrophage polarization during early pregnancy? SUMMARY ANSWER: PD-1 signaling is a major regulator of macrophage differentiation and function, and it is critical for the success of a pregnancy. WHAT IS KNOWN ALREADY: The predominance of decidual macrophages (DMs) with an M2 phenotype is an important contributor to maternal-fetal tolerance during early pregnancy. STUDY DESIGN, SIZE, DURATION: Twenty-four women with recurrent miscarriage (RM) and 70 women undergoing elective termination of an early normal pregnancy (NP) were included. Twelve female CBA/J, four male DBA/2, and four male BALB/c mice were included and mating carried out. The 12 CBA/J pregnant mice were then categorized into three groups of four mice: healthy control group CBA/J×BALB/c, abortion-prone pregnant group CBA/J×DBA/2 and normal pregnancies CBA/J×BALB/c treated with anti-PD-1 monoclonal antibodies. PARTICIPANTS/MATERIALS, SETTING, METHODS: The profile of DMs, and the expression of PD-1 and PD-L1 in DMs from women with NP and RM were measured by flow cytometry. PD-L1 expression in human villi was determined by quantitative RT-PCR (qRT-PCR) and western blot. An in vitro model consisting of peripheral CD14+ monocytes isolated from women with NP was used. The profile of differentiated macrophages and their phagocytotic activity were then measured by flow cytometry. The mRNA levels of genes potentially underlying macrophage polarization modulated by PD-1 signaling were determined by qRT-PCR. Twelve pregnant mice were included in our in vivo model and underwent different treatment. The embryo resorption rate, and macrophage profile as well as PD-1 expression in murine spleens and uterus were analyzed by flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with NP, women with RM had elevated percentages of M1 DMs (P < 0.01), and reduced frequencies of M2 DMs (P < 0.05), as well as decreased PD-1 protein expression (P < 0.05) in the DMs. In addition, decreased mRNA and protein levels of PD-L1 expression in placental villi were observed in women with RM (P < 0.001). Using in vitro experiments, compared to the control group, we found that PD-1 activation by recombinant human (rh) PD-L1 Fc (human PD-L1 fused to the Fc region of human IgG1) drove the differentiation of macrophages with immuno-modulatory characteristics (P < 0.01). However, PD-1 blockade promoted dominance of the M1 phenotype (P < 0.01). PD-1 polarized macrophages showed enhanced phagocytic activity (P < 0.01), which was decreased with PD-1 blockade (P < 0.001). Furthermore, PD-1 blockade promoted the expression of pro-inflammatory cytokines and interferon regulatory factor (IRF) 5 (P < 0.05), while IRF4 expression was inhibited (P < 0.05). In addition, PD-1 blockade promoted macrophage glycolysis (P < 0.01) and inhibited fatty acid oxidation (P < 0.05). The mRNA expression levels of both phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin and mitogen-activated protein kinase/extracellular signal-regulated kinase/extracellular signal-regulated kinase were upregulated (P < 0.05) with PD-1 blockade during DM metabolic reprogramming. Moreover, in vivo mice data showed that PD-1 blockade or deficiency was associated with decreased M2 percentages at the maternal-fetal interface (P < 0.05) and embryo loss (P < 0.05). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Whether the changes in DM polarization seen in miscarriage tissues are a cause or consequence of the demise of the pregnancy still requires further investigation. In addition, conducting metabolite analysis is required to further measure bioenergetic profiles. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study on the role of the PD-1/PD-L1 axis in macrophage polarization during early pregnancy; such exploration enhances our understanding of the physiology of early pregnancy. Our study also indicates that targeting the PD-1 pathway may represent a novel therapeutic strategy to prevent pregnancy loss. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Nature Science Foundation of China (No. 81671490) and Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College, HUST (No. 5001519002). None of the authors has any conflict of interest to declare.

Cutting Edge: Fetal/Placental Type I IFN Can Affect Maternal Survival and Fetal Viral Load during Viral Infection.

Pregnant women have greater mortality and complications associated with viral infections compared with the general population, but the reason for the increased susceptibility is not well defined. Placenta type I IFN is an important immune modulator and protects the pregnancy. We hypothesized that loss of placental IFN affects the regulation of the maternal immune system, resulting in the differential response to infections observed in pregnancy. Pregnant mice lacking the IFN-α/ß receptor (IFNAR) became viremic and had higher mortality compared with nonpregnant animals. Notably, an embryo with functional IFN signaling alone was sufficient to rescue the pregnant IFNAR-/- dam from virus-associated demise. Placental IFN was also an important regulator of viral replication in placental tissue and significantly affected viral transmission to the fetus. These findings highlight the role of fetal/placental IFN in the modulation of viral infection in the mother and fetus.

Viral Infection Sensitizes Human Fetal Membranes to Bacterial Lipopolysaccharide by MERTK Inhibition and Inflammasome Activation.

Chorioamnionitis, premature rupture of fetal membranes (FMs), and subsequent preterm birth are associated with local infection and inflammation, particularly IL-1ß production. Although bacterial infections are commonly identified, other microorganisms may play a role in the pathogenesis. Because viral pandemics, such as influenza, Ebola, and Zika, are becoming more common, and pregnant women are at increased risk for associated complications, this study evaluated the impact that viral infection had on human FM innate immune responses. This study shows that a herpes viral infection of FMs sensitizes the tissue to low levels of bacterial LPS, giving rise to an exaggerated IL-1ß response. Using an ex vivo human FM explant system and an in vivo mouse model of pregnancy, we report that the mechanism by which this aggravated inflammation arises is through the inhibition of the TAM receptor, MERTK, and activation of the inflammasome. The TAM receptor ligand, growth arrest specific 6, re-establishes the normal FM response to LPS by restoring and augmenting TAM receptor and ligand expression, as well as by preventing the exacerbated IL-1ß processing and secretion. These findings indicate a novel mechanism by which viruses alter normal FM immune responses to bacteria, potentially giving rise to adverse pregnancy outcomes.

Adipocyte microenvironment promotes Bclxl expression and confers chemoresistance in ovarian cancer cells.

Resistance to mitochondria-initiated apoptosis is a hallmark of chemoresistant cancer stem cells including CD44+/MyD88+ epithelial ovarian cancer (EOC) stem cells. This is controlled by members of the Bcl2 family of proteins, which function as rheostats of mitochondrial stability. We observed a differential expression profile of Bcl2 family members comparing the chemoresistant EOC stem cells and the chemosensitive CD44-/MyD88- EOC cells. Chemoresistant EOC stem cells surprisingly express higher levels of the pro-apoptotic members Bak and Bax compared to the chemosensitive EOC cells. In addition, whereas chemosensitive EOC cells preferentially express Bcl2, chemoresistant EOC stem cells preferentially express Bclxl. In the EOC stem cells, 40% knock-down of Bclxl expression was sufficient to induce the full activation of caspases and this can be reversed by concurrent knock-down of Puma. More importantly, we demonstrate that Bclxl expression levels in EOC cells is dynamic and can be regulated by microenvironments that are enriched with the pro-inflammatory cytokine IL-6 such as the cancer stem cell and adipocyte niches. Adipocyte-induced upregulation of Bclxl correlated with acquisition of chemoresistance and thus demonstrates how a specific microenvironment can regulate the expression of apoptotic proteins and confer chemoresistance.