Effector memory CD8 T cell response elicits Hepatitis E Virus genotype 3 pathogenesis in the elderly.

Genotype 3 Hepatitis E virus (HEV-3) is an emerging threat for aging population. More than one third of older infected patients develops clinical symptoms with severe liver damage, while others remain asymptomatic. The origin of this discrepancy is still elusive although HEV-3 pathogenesis appears to be immune-mediated. Therefore, we investigated the role of CD8 T cells in the outcome of the infection in immunocompetent elderly subjects. We enrolled twenty two HEV-3-infected patients displaying similar viral determinants and fifteen healthy donors. Among the infected group, sixteen patients experienced clinical symptoms related to liver disease while six remained asymptomatic. Here we report that symptomatic infection is characterized by an expansion of highly activated effector memory CD8 T (EM) cells, regardless of antigen specificity. This robust activation is associated with key features of early T cell exhaustion including a loss in polyfunctional type-1 cytokine production and partial commitment to type-2 cells. In addition, we show that bystander activation of EM cells seems to be dependent on the inflammatory cytokines IL-15 and IL-18, and is supported by an upregulation of the activating receptor NKG2D and an exuberant expression of T-Bet and T-Bet-regulated genes including granzyme B and CXCR3. We also show that the inflammatory chemokines CXCL9-10 are increased in symptomatic patients thereby fostering the recruitment of highly cytotoxic EM cells into the liver in a CXCR3-dependent manner. Finally, we find that the EM-biased immune response returns to homeostasis following viral clearance and disease resolution, further linking the EM cells response to viral burden. Conversely, asymptomatic patients are endowed with low-to-moderate EM cell response. In summary, our findings define immune correlates that contribute to HEV-3 pathogenesis and emphasize the central role of EM cells in governing the outcome of the infection.

Stem Cells-Derived Extracellular Vesicles: Potential Therapeutics for Wound Healing in Chronic Inflammatory Skin Diseases.

Endosome-derived small extracellular vesicles (EVs), often referred to as exosomes, are produced by almost all, if not all, cell types, and are critical for intercellular communication. They are composed of a lipid bilayer associated with membrane proteins and contain a payload of lipids, proteins and regulatory RNAs that depends on the parental cell physiological condition. By transferring their "cargo", exosomes can modulate the phenotype of neighboring and distant cells. Stem cells (SC) were widely studied for therapeutic applications regarding their regenerative/reparative potential as well as their immunomodulatory properties. Whether from autologous or allogeneic source, SC beneficial effects in terms of repair and regeneration are largely attributed to their paracrine signaling notably through secreted EVs. Subsequently, SC-derived EVs have been investigated for the treatment of various diseases, including inflammatory skin disorders, and are today fast-track cell-free tools for regenerative/reparative strategies. Yet, their clinical application is still facing considerable challenges, including production and isolation procedures, and optimal cell source. Within the emerging concept of "allogeneic-driven benefit" for SC-based therapies, the use of EVs from allogeneic sources becomes the pragmatic choice although a universal allogeneic cell source is still needed. As a unique temporary organ that ensures the mutual coexistence of two allogeneic organisms, mother and fetus, the human placenta offers a persuasive allogeneic stem cell source for development of therapeutic EVs. Advancing cell-free therapeutics nurtures great hope and provides new perspectives for the development of safe and effective treatment in regenerative/reparative medicine and beyond. We will outline the current state of the art in regard of EVs, summarize their therapeutic potential in the context of skin inflammatory disorders, and discuss their translational advantages and hurdles.

A CRISPR Activation Screen Identifies Genes That Protect against Zika Virus Infection.

Zika virus (ZIKV) is an arthropod-borne emerging pathogen causing febrile illness. ZIKV is associated Guillain-Barré syndrome and other neurological complications. Infection during pregnancy is associated with pregnancy complications and developmental and neurological abnormalities collectively defined as congenital Zika syndrome. There is still no vaccine or specific treatment for ZIKV infection. To identify host factors that can rescue cells from ZIKV infection, we used a genome-scale CRISPR activation screen. Our highly ranking hits included a short list of interferon-stimulated genes (ISGs) previously reported to have antiviral activity. Validation of the screen results highlighted interferon lambda 2 (IFN-λ2) and interferon alpha-inducible protein 6 (IFI6) as genes providing high levels of protection from ZIKV. Activation of these genes had an effect on an early stage in viral infection. In addition, infected cells expressing single guide RNAs (sgRNAs) for both of these genes displayed lower levels of cell death than did the controls. Furthermore, the identified genes were significantly induced in ZIKV-infected placenta explants. Thus, these results highlight a set of ISGs directly relevant for rescuing cells from ZIKV infection or its associated cell death and substantiate CRISPR activation screens as a tool to identify host factors impeding pathogen infection.IMPORTANCE Zika virus (ZIKV) is an emerging vector-borne pathogen causing a febrile disease. ZIKV infection might also trigger Guillain-Barré syndrome, neuropathy, and myelitis. Vertical transmission of ZIKV can cause fetus demise, stillbirth, or severe congenital abnormalities and neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We used a genome-wide CRISPR activation screen, where genes are activated from their native promoters to identify host cell factors that protect cells from ZIKV infection or associated cell death. The results provide a better understanding of key host factors that protect cells from ZIKV infection and might assist in identifying novel antiviral targets.

Peripheral Plasma and Semen Cytokine Response to Zika Virus in Humans.

We assessed Zika virus RNA and select cytokine levels in semen, blood, and plasma samples from an infected patient in South America. Viral RNA was detected in semen >2 months after viremia clearance; cytokine profiles differed in semen and plasma. After viremia, Zika virus appears to become compartmentalized in the male reproductive tract.

[Hepatitis E virus: from the infected organism to the cellular response]. / Virus de l'hépatite E : de l'organisme infecté à la réponse cellulaire.

Hepatitis E virus (HEV) presents a worldwide distribution. In developing countries, hepatitis E, related to HEV1 and HEV2, is a waterborne disease. In developed countries, hepatitis E is a zoonotic disease due to HEV3 and HEV4. It is mainly transmitted through meat consumption from animal reservoirs such as pig, boar, deer and rabbit. New clinical forms include neurological manifestations that are now clearly associated with HEV3 infection. Recent studies showed that ORF1 polyprotein was able to disrupt the innate immune response. It was also shown that ORF2 protein exists at least in two forms: a free, glycosylated form and a non-glycosylated form, which assembles to form the capsid. Lastly, it was shown that ORF3 protein, involved in the virus egress, acts as a viroporin. New culture systems and animal models have been developed recently, and will be very helpful to complete our understanding of HEV life cycle and pathogenesis.

Human cytomegalovirus infection elicits new decidual natural killer cell effector functions.

During the first trimester of pregnancy the uterus is massively infiltrated by decidual natural killer cells (dNK). These cells are not killers, but they rather provide a microenvironment that is propitious to healthy placentation. Human cytomegalovirus (HCMV) is the most common cause of intrauterine viral infections and a known cause of severe birth defects or fetal death. The rate of HCMV congenital infection is often low in the first trimester of pregnancy. The mechanisms controlling HCMV spreading during pregnancy are not yet fully revealed, but evidence indicating that the innate immune system plays a role in controlling HCMV infection in healthy adults exists. In this study, we investigated whether dNK cells could be involved in controlling viral spreading and in protecting the fetus against congenital HCMV infection. We found that freshly isolated dNK cells acquire major functional and phenotypic changes when they are exposed to HCMV-infected decidual autologous fibroblasts. Functional studies revealed that dNK cells, which are mainly cytokines and chemokines producers during normal pregnancy, become cytotoxic effectors upon their exposure to HCMV-infected autologous decidual fibroblasts. Both the NKG2D and the CD94/NKG2C or 2E activating receptors are involved in the acquired cytotoxic function. Moreover, we demonstrate that CD56(pos) dNK cells are able to infiltrate HCMV-infected trophoblast organ culture ex-vivo and to co-localize with infected cells in situ in HCMV-infected placenta. Taken together, our results present the first evidence suggesting the involvement of dNK cells in controlling HCMV intrauterine infection and provide insights into the mechanisms through which these cells may operate to limit the spreading of viral infection to fetal tissues.

The up side of decidual natural killer cells: new developments in immunology of pregnancy.

Early phases of human pregnancy are associated with the accumulation of a unique subset of natural killer (NK) cells in the maternal decidua. Decidual NK (dNK) cells that are devoid of cytotoxicity play a pivotal role in successful pregnancy. By secreting large amounts of cytokines/chemokines and angiogenic factors, dNK cells participate in all steps of placentation including trophoblast invasion into the maternal endometrium and vascular remodelling. In this review, we summarize some of dNK cell features and discuss more recent exciting data that challenge the conventional view of these cells. Our new data demonstrate that dNK cells undergo fine tuning or even subvert their classical inhibitory machinery and turn into a real defence force in order to prevent the spread of viruses to fetal tissue. Today it is not clear how these phenotypic and functional adaptations impact cellular cross-talk at the fetal-maternal interface and tissue homeostasis. Ultimately, precise understanding of the molecular mechanisms that govern dNK cell plasticity during congenital human cytomegalovirus infection should lead to the design of more robust strategies to reverse immune escape during viral infection and cancer.

Plasma Level of ATPase Inhibitory Factor 1 and Intrinsic Capacity in Community-Dwelling Older Adults: Prospective Data From the MAPT Study.

BACKGROUND: Intrinsic capacity (IC) is a concept related to functionality that reflects healthy aging. ATPase inhibitory factor 1 (IF1) is a multifaceted protein that regulates mitochondrial oxidative phosphorylation (OXPHOS), and may be involved in IC. The objective of this study is to investigate the association between plasma levels of IF1 and IC changes in community-dwelling older adults. METHODS: Community-dwelling older adults from the Multidomain Alzheimer Preventive Trial (MAPT Study) were enrolled in this study. A composite IC score was calculated based on 4 IC domains: locomotion, psychological dimension, cognition, and vitality (with data available annually over 4 years of follow-up). Secondary analyses were conducted on the sensory domain (with data available only for 1 year of follow-up). Mixed-model linear regression adjusted for confounders was conducted. RESULTS: A total of 1 090 participants with usable IF1 values were included in the study (75.3 ± 4.4 years; 64% females). Compared to the lowest quartile, both the low- and high-intermediate IF1 quartiles were found to be cross-sectionally associated with greater composite IC scores across 4 domains (ßlow-intermediate, 1.33; 95% confidence interval [CI] 0.06-2.60 and ßhigh-intermediate, 1.78; 95% CI 0.49-3.06). In the secondary analyses, the highest quartile was found to be associated with a slower decline in composite IC scores across 5 domains over 1 year (ßhigh 1.60; 95% CI 0.06-3.15). The low- and high-intermediate IF1 quartiles were also found to be cross-sectionally associated with greater locomotion (ßlow-intermediate, 2.72; 95% CI 0.36-5.08) and vitality scores (ßhigh-intermediate, 1.59; 95% CI 0.06-3.12), respectively. CONCLUSIONS: This study is the first to demonstrate that levels of circulating IF1, a mitochondrial-related biomarker, are associated with IC composite scores in both cross-sectional and prospective analyses among community-dwelling older adults. However, further research is needed to confirm these findings and elucidate the potential underlying mechanisms that may explain these associations.

Productive HIV-1 infection of tissue macrophages by fusion with infected CD4+ T cells.

Macrophages are essential for HIV-1 pathogenesis and represent major viral reservoirs. Therefore, it is critical to understand macrophage infection, especially in tissue macrophages, which are widely infected in vivo, but poorly permissive to cell-free infection. Although cell-to-cell transmission of HIV-1 is a determinant mode of macrophage infection in vivo, how HIV-1 transfers toward macrophages remains elusive. Here, we demonstrate that fusion of infected CD4+ T lymphocytes with human macrophages leads to their efficient and productive infection. Importantly, several tissue macrophage populations undergo this heterotypic cell fusion, including synovial, placental, lung alveolar, and tonsil macrophages. We also find that this mode of infection is modulated by the macrophage polarization state. This fusion process engages a specific short-lived adhesion structure and is controlled by the CD81 tetraspanin, which activates RhoA/ROCK-dependent actomyosin contractility in macrophages. Our study provides important insights into the mechanisms underlying infection of tissue-resident macrophages, and establishment of persistent cellular reservoirs in patients.

Decidua Basalis: An Ex Vivo Model to Study HIV-1 Infection During Pregnancy and Beyond.

The human decidua basalis, main uterine mucosa during pregnancy, provides an ex vivo model for studying natural protection of macrophages against HIV-1 infection at the mucosal level. Beyond pregnancy, the decidua constitutes also a valuable tool to assess tissue-resident macrophage infection. Here, we provide a detailed protocol for decidual macrophage purification and tissue infection.

The mechanisms underlying the immune control of Zika virus infection at the maternal-fetal interface.

Unlike other Flaviviruses, Zika virus (ZIKV) infection during the first trimester of pregnancy causes severe pregnancy outcomes including the devastating microcephaly and diseases associated with placental dysfunctions. We have previously reported that the maternal decidua basalis, the major maternal-fetal interface, serves as a replication platform enabling virus amplification before dissemination to the fetal compartment. However, the rate of congenital infection is quite low, suggesting the presence of a natural barrier against viral infection. Using primary cells from first-trimester pregnancy samples, we investigated in this study how the maternal decidua can interfere with ZIKV infection. Our study reveals that whether through their interactions with dNK cells, the main immune cell population of the first-trimester decidua, or their production of proinflammatory cytokines, decidual stromal cells (DSCs) are the main regulators of ZIKV infection during pregnancy. We also validate the functional role of AXL as a crucial receptor for ZIKV entry in DSCs and demonstrate that targeted inhibition of ligand-receptor interaction at the early stage of the infection is effective in drastically reducing virus pathogenesis at the maternal-fetal interface. Collectively, our results provide insights into the mechanisms through which ZIKV infection and spreading can be limited. The strategy of circumventing viral entry at the maternal-fetus interface limits virus dissemination to fetal tissues, thereby preventing congenital abnormalities.

Innate Immune Response to Viral Infections at the Maternal-Fetal Interface in Human Pregnancy.

The placenta, the first and largest organ to develop after conception, not only nurtures and promotes the development of the conceptus, but, it also functions as a barrier against invading pathogens. Early phases of pregnancy are associated with expansion of specific subsets of Natural Killer cells (dNK) and macrophages (dMφ) at the maternal uterine mucosa, the basal decidua. In concert with cells of fetal origin, dNK cells, and dMφ orchestrate all steps of placenta and fetus development, and provide the first line of defense to limit vertical transmission. However, some pathogens that infect the mother can overcome this protective barrier and jeopardize the fetus health. In this review, we will discuss how members of the classical TORCH family (Toxoplasma, Other, Rubella, Cytomegalovirus, and Herpes simplex virus) and some emerging viruses (Hepatitis E virus, Zika virus, and SARS-CoV2) can afford access to the placental fortress. We will also discuss how changes in the intrauterine environment as a consequence of maternal immune cell activation contribute to placental diseases and devastating pregnancy outcomes.

Critical and differential roles of NKp46- and NKp30-activating receptors expressed by uterine NK cells in early pregnancy.

In early human pregnancy, uterine decidual NK cells (dNK) are abundant and considered as cytokine producers but poorly cytotoxic despite their cytolytic granule content, suggesting a negative control of this latter effector function. To investigate the basis of this control, we examined the relative contribution to the cytotoxic function of different activating receptors expressed by dNK. Using a multicolor flow cytometry analysis, we found that freshly isolated dNK exhibit a unique repertoire of activating and inhibitory receptors, identical among all the donors tested. We then demonstrated that in fresh dNK, mAb-specific engagement of NKp46-, and to a lesser extent NKG2C-, but not NKp30-activating receptors induced intracellular calcium mobilization, perforin polarization, granule exocytosis and efficient target cell lysis. NKp46-mediated cytotoxicity is coactivated by CD2 but dramatically blocked by NKG2A coengagement, indicating that the dNK cytotoxic potential could be tightly controlled in vivo. We finally found that in dNK, mAb-specific engagement of NKp30, but not NKp46, triggered the production of IFN-gamma, TNF-alpha, MIP-1alpha, MIP-1beta, and GM-CSF proinflammatory molecules. These data demonstrate a differential, controlled role of NKp46- and NKp30-activating receptors expressed by dNK that could be critical for the outcome of pregnancy and the killing of uterine cells infected by pathogens.

Zika Virus Targets Multiple Tissues and Cell Types During the First Trimester of Pregnancy.

The 2016 Zika virus (ZIKV) outbreak in the Americas has been characterized by an increased association frequency of fetal neuropathological abnormalities. To have a comprehensive and accurate knowledge of key elements of the clinically observed neurologic dysfunctions in Zika-infected babies, ZIKV transmission from mother to fetus needs to be deeply studied. Thus, it is important to determine the role of both virus-targeted cells and tissues within the mother-fetus interface. Cellular tropism and mechanisms of ZIKV transmission from mother to the fetus during early pregnancy still remain unknown on many aspects. To improve the characterization of the maternal-fetal ZIKV transmission, we have set up an ex vivo model using an organ culture approach with a light-invasive sampling from the first trimester of pregnancy samples. Thus, here we provide evidence that circulating epidemic ZIKV strains from Latin America widely target and destroy reproductive tissues, including the decidua basalis, fetal placenta, and umbilical cord. In addition, we show that ZIKV is able to differentially replicate in a large range of both maternal and fetal cells, including decidual fibroblasts and macrophages, fetal trophoblast and Hofbauer cells, as well as umbilical cord mesenchymal stem cells. This primary and broad ZIKV cellular tropism and the resulting abundant cytopathic-induced tissue effects during the first trimester of pregnancy show the upstream path of clinically observed congenital damages.

Metabolic reprogramming by Zika virus provokes inflammation in human placenta.

The recent outbreak of Zika virus (ZIKV) was associated with birth defects and pregnancy loss when maternal infection occurs in early pregnancy, but specific mechanisms driving placental insufficiency and subsequent ZIKV-mediated pathogenesis remain unclear. Here we show, using large scale metabolomics, that ZIKV infection reprograms placental lipidome by impairing the lipogenesis pathways. ZIKV-induced metabolic alterations provide building blocks for lipid droplet biogenesis and intracellular membrane rearrangements to support viral replication. Furthermore, lipidome reprogramming by ZIKV is paralleled by the mitochondrial dysfunction and inflammatory immune imbalance, which contribute to placental damage. In addition, we demonstrate the efficacy of a commercially available inhibitor in limiting ZIKV infection, provides a proof-of-concept for blocking congenital infection by targeting metabolic pathways. Collectively, our study provides mechanistic insights on how ZIKV targets essential hubs of the lipid metabolism that may lead to placental dysfunction and loss of barrier function.

Features of Human Decidual NK Cells in Healthy Pregnancy and During Viral Infection.

The hallmark of human early pregnancy is the accumulation of a unique population of Natural Killer (dNK) cells at the main maternal-fetal interface, the decidua basalis. dNK cells play a crucial role in successful placentation probably by orchestrating the invasion of trophoblast cells deep into the decidua basalis and remodeling of the maternal spiral arteries. Recent advances in the field emphasize the importance of the local microenvironment in shaping both the phenotype and the effector functions of these innate lymphoid cells. Despite slow progress in the field, ex vivo studies revealed that dNK cells sense and destroy infected cells in order to protect the fetus from invading pathogens. In this review, we will discuss key features of dNK cells during healthy pregnancy as well as their functional adaptations in limiting pathogen dissemination to the growing conceptus. The challenge is to better understand the plasticity of dNK cells in the maternal-fetal interface. Such insights would enable greater understanding of the pathogenesis in congenital infections and pregnancy disorders.

Extracellular Vesicles Released by Allogeneic Human Cardiac Stem/Progenitor Cells as Part of Their Therapeutic Benefit.

The positive effects of therapeutic human allogeneic cardiac stem/progenitor cells (hCPC) in terms of cardiac repair/regeneration are very likely mediated by paracrine effects. Our previous studies revealed the advantageous immune interactions of allogeneic hCPC and proposed them as part of the positive paracrine effects occurring upon their application postmyocardial infarction (MI). Currently, extracellular vesicles/exosomes (EV/Exs) released by stem/progenitor cells are also proposed as major mediators of paracrine effects of therapeutic cells. Along this line, we evaluated contribution of EV/Exs released by therapeutic hCPC to the benefit of their successful allogeneic clinical application. Through tailored allogeneic in vitro human assay models mimicking the clinical setting, we demonstrate that hCPC-released EV/Exs were rapidly and efficiently up-taken by chief cellular actors of cardiac repair/regeneration. This promoted MAPK/Erk1/2 activation, migration, and proliferation of human leukocyte antigens (HLA)-mismatched hCPC, mimicking endogenous progenitor cells and cardiomyocytes, and enhanced endothelial cell migration, growth, and organization into tube-like structures through activation of several signaling pathways. EV/Exs also acted as pro-survival stimuli for HLA-mismatched monocytes tuning their phenotype toward an intermediate anti-inflammatory pro-angiogenic phenotype. Thus, while positively impacting the intrinsic regenerative and angiogenic programs, EV/Exs released by therapeutic allogeneic hCPC can also actively contribute to shaping MI-inflammatory environment, which could strengthen the benefits of hCPC allogeneic interactions. Collectively, our data might forecast the application of allogeneic hCPC followed by their cell-free EV/Exs as a strategy that will not only elicit the cell-contact mediated reparative/regenerative immune response but also have the desired long-lasting effects through the EV/Exs. Stem Cells Translational Medicine 2019;8:911&924.

Genotype specific pathogenicity of hepatitis E virus at the human maternal-fetal interface.

Hepatitis E virus (HEV) infection, particularly HEV genotype 1 (HEV-1), can result in fulminant hepatic failure and severe placental diseases, but mechanisms underlying genotype-specific pathogenicity are unclear and appropriate models are lacking. Here, we model HEV-1 infection ex vivo at the maternal-fetal interface using the decidua basalis and fetal placenta, and compare its effects to the less-pathogenic genotype 3 (HEV-3). We demonstrate that HEV-1 replicates more efficiently than HEV-3 both in tissue explants and stromal cells, produces more infectious progeny virions and causes severe tissue alterations. HEV-1 infection dysregulates the secretion of several soluble factors. These alterations to the cytokine microenvironment correlate with viral load and contribute to the tissue damage. Collectively, this study characterizes an ex vivo model for HEV infection and provides insights into HEV-1 pathogenesis during pregnancy that are linked to high viral replication, alteration of the local secretome and induction of tissue injuries.

Major histocompatibility complex class II transcriptional platform: assembly of nuclear factor Y and regulatory factor X (RFX) on DNA requires RFX5 dimers.

Major histocompatibility complex class II (MHC-II) genes are regulated in a B-cell-specific and gamma interferon-inducible manner. Conserved upstream sequences (CUS) in their compact promoters bind nuclear factor Y (NFY) and regulatory factor X (RFX) complexes. These DNA-bound proteins form a platform that attracts the class II transactivator, which initiates and elongates MHC-II transcription. In this report, we analyzed the complex assembly of these DNA-bound proteins. First, we found that NFY can interact with RFX in cells. In particular, NFYA and NFYC bound RFXANK/B in vitro. Next, RFX5 formed dimers in vivo and in vitro. Within a leucine-rich stretch N-terminal to the DNA-binding domain in RFX5, the leucine at position 66 was found to be critical for this self-association. Mutant RFX5 proteins that could not form dimers also did not support the formation of higher-order DNA-protein complexes on CUS in vitro or MHC-II transcription in vivo. We conclude that the MHC-II transcriptional platform begins to assemble off CUS and then binds DNA via multiple, spatially constrained interactions. These findings offer one explanation of why in the Bare Lymphocyte Syndrome, which is a congenital severe combined immunodeficiency, MHC-II promoters are bare when any subunit of RFX is mutated or missing.