Mouse Trophoblast Cells Have Attenuated Responses to TNF-α and IFN-γ and Can Avoid Synergic Cytotoxicity of the Two Cytokines.

TNF-α and IFN-γ are two inflammatory cytokines that play critical roles in immune responses, but they can also negatively affect cell proliferation and viability. In particular, the combination of the two cytokines (TNF-α/IFN-γ) synergistically causes cytotoxicity in many cell types. We recently reported that mouse embryonic stem cells (ESCs) isolated from the blastocyst stage embryo do not respond to TNF-α and have limited response to IFN-γ, thereby avoiding TNF-α/IFN-γ cytotoxicity. The current study expanded our investigation to mouse trophoblast stem cells (TSCs) and their differentiated trophoblasts (TSC-TBs), the precursors and the differentiated cells of the placenta, respectively. In this study, we report that the combination of TNF-α/IFN-γ does not show the cytotoxicity to TSCs and TSC-TBs that otherwise effectively kills fibroblasts, similar to ESCs. Although ESCs, TSCs, and TSC-TBs are dramatically different in their growth rate, morphology, and physiological functions, they nevertheless share a similarity in being able to avoid TNF-α/IFN-γ cytotoxicity. We propose that this unique immune property may serve as a protective mechanism that limits cytokine cytotoxicity in the blastocyst. With molecular and cellular approaches and genome-wide transcriptomic analysis, we have demonstrated that the attenuated NF-κB and STAT1 transcription activation is a limiting factor that restricts the effect of TNF-α/IFN-γ on TSCs and TSC-TBs.

Post-transcriptional capping generates coenzyme A-linked RNA.

NAD can be inserted co-transcriptionally via non-canonical initiation to form NAD-RNA. However, that mechanism is unlikely for CoA-linked RNAs due to low intracellular concentration of the required initiator nucleotide, 3'-dephospho-CoA (dpCoA). We report here that phosphopantetheine adenylyltransferase (PPAT), an enzyme of CoA biosynthetic pathway, accepts RNA transcripts as its acceptor substrate and transfers 4'-phosphopantetheine to yield CoA-RNA post-transcriptionally. Synthetic natural (RNAI) and small artificial RNAs were used to identify the features of RNA that are needed for it to serve as PPAT substrate. RNAs with 4-10 unpaired nucleotides at the 5' terminus served as PPAT substrates, but RNAs having <4 unpaired nucleotides did not undergo capping. No capping was observed when the +1A was changed to G or when 5' triphosphate was removed by RNA pyrophosphohydrolase (RppH), suggesting the enzyme recognizes pppA-RNA as an ATP analog. PPAT binding affinities were equivalent for transcripts with +1A, +1 G, or 5'OH (+1A), indicating that productive enzymatic recognition is driven more by local positioning effects than by overall binding affinity. Capping rates were independent of the number of unpaired nucleotides in the range of 4-10 nucleotides. Capping was strongly inhibited by ATP, reducing CoA-RNA production ~70% when equimolar ATP and substrate RNA were present. Dual bacterial expression of candidate RNAs with different 5' structures followed by CoA-RNA CaptureSeq revealed 12-fold enrichment of the better PPAT substrate, consistent with in vivo CoA-capping of RNA transcripts by PPAT. These results suggest post-transcriptional RNA capping as a possible mechanism for the biogenesis of CoA-RNAs in bacteria.

Mouse Trophoblast Cells Can Provide IFN-Based Antiviral Protection to Embryonic Stem Cells via Paracrine Signaling.

The blastocyst is the preimplantation stage embryo that consists of two major components: the inner cell mass (ICM) and the trophectoderm (TE). The ICM gives rise to the fetus and some extraembryonic tissues whereas the TE contributes to development of the placenta. Previous studies have demonstrated that both human and mouse embryonic stem cells (ESCs) derived from the ICM are deficient in expressing type I IFNs in response to viral infection. In this study, we investigated the IFN response in mouse trophoblast stem cells (TSCs) and their in vitro differentiated trophoblasts (TSC-TBs). In this study, we report that, unlike ESCs, TSCs have a functional IFN system. They can express type I IFNs in response to viral stimuli and express IFN-stimulated genes in response to type I IFNs. TSC-TBs have a further developed IFN system and acquired the ability to express specialized type III IFN-λ. Furthermore, TSCs and TSC-TBs can provide ESCs with antiviral activity against Chikungunya, West Nile, and Zika virus infection, as demonstrated with a novel coculture model that simulates the temporal and spatial relationship between the ICM and the TE in a blastocyst. Taken together, our data demonstrate that mouse ESCs can respond to type I IFNs and gain IFN-based antiviral protection from TSCs and TSC-TBs via paracrine signaling mechanisms even though they themselves are unable to express type I IFNs.

Dicer and PKR as Novel Regulators of Embryonic Stem Cell Fate and Antiviral Innate Immunity.

Embryonic stem cells (ESCs) represent a unique cell population in the blastocyst stage embryo. They have been intensively studied as a promising cell source for regenerative medicine. Recent studies have revealed that both human and mouse ESCs are deficient in expressing IFNs and have attenuated inflammatory responses. Apparently, the ability to express IFNs and respond to certain inflammatory cytokines is not "innate" to ESCs but rather is developmentally acquired by somatic cells during differentiation. Accumulating evidence supports a hypothesis that the attenuated innate immune response may serve as a protective mechanism allowing ESCs to avoid immunological cytotoxicity. This review describes our current understanding of the molecular basis that shapes the immune properties of ESCs. We highlight the recent findings on Dicer and dsRNA-activated protein kinase R as novel regulators of ESC fate and antiviral immunity and discuss how ESCs use alternative mechanisms to accommodate their stem cell properties.

Murine Trophoblast Stem Cells and Their Differentiated Cells Attenuate Zika Virus In Vitro by Reducing Glycosylation of the Viral Envelope Protein.

Zika virus (ZIKV) infection during pregnancy can cause devastating fetal neuropathological abnormalities, including microcephaly. Most studies of ZIKV infection in pregnancy have focused on post-implantation stage embryos. Currently, we have limited knowledge about how a pre-implantation stage embryo deals with a viral infection. This study investigates ZIKV infection on mouse trophoblast stem cells (TSCs) and their in vitro differentiated TSCs (DTSCs), which resemble the cellular components of the trophectoderm layer of the blastocyst that later develops into the placenta. We demonstrate that TSCs and DTSCs are permissive to ZIKV infection; however, ZIKV propagated in TSCs and DTSCs exhibit substantially lower infectivity, as shown in vitro and in a mouse model compared to ZIKV that was generated in Vero cells or mouse embryonic fibroblasts (MEFs). We further show that the low infectivity of ZIKV propagated in TSCs and DTSCs is associated with a reduced level of glycosylation on the viral envelope (E) proteins, which are essential for ZIKV to establish initial attachment by binding to cell surface glycosaminoglycans (GAGs). The decreased level of glycosylation on ZIKV E is, at least, partially due to the low-level expression of a glycosylation-related gene, Hexa, in TSCs and DTSCs. Furthermore, this finding is not limited to ZIKV since similar observations have been made as to the chikungunya virus (CHIKV) and West Nile virus (WNV) propagated in TSCs and DTSCs. In conclusion, our results reveal a novel phenomenon suggesting that murine TSCs and their differentiated cells may have adapted a cellular glycosylation system that can limit viral infectivity by altering the glycosylation of viral envelope proteins, therefore serving as a unique, innate anti-viral mechanism in the pre-implantation stage embryo.

Dicer represses the interferon response and the double-stranded RNA-activated protein kinase pathway in mouse embryonic stem cells.

Recent studies have demonstrated that embryonic stem cells (ESCs) are deficient in expressing type I interferons (IFN), the cytokines that play key roles in antiviral responses. However, the underlying molecular mechanisms and biological implications of this finding are poorly understood. In this study, we developed a synthetic RNA-based assay that can simultaneously assess multiple forms of antiviral responses. Dicer is an enzyme essential for RNA interference (RNAi), which is used as a major antiviral mechanism in invertebrates. RNAi activity is detected in wild-type ESCs but is abolished in Dicer knockout ESCs (D-/-ESCs) as expected. Surprisingly, D-/-ESCs have gained the ability to express IFN, which is otherwise deficient in wild-type ESCs. Furthermore, D-/-ESCs have constitutively active double-stranded RNA (dsRNA)-activated protein kinase (PKR), an enzyme that is also involved in antiviral response. D-/-ESCs show increased sensitivity to the cytotoxicity resulting from RNA transfection. The effects of dsRNA can be partly replicated with a synthetic B2RNA corresponding to the retrotransposon B2 short interspersed nuclear element. B2RNA has secondary structure features of dsRNA and accumulates in D-/-ESCs, suggesting that B2RNA could be a cellular RNA that activates PKR and contributes to the decreased cell proliferation and viability of D-/-ESCs. Treatment of D-/-ESCs with a PKR inhibitor and IFNß-neutralizing antibodies increased cell proliferation rate and cell viability. Based on these findings, we propose that, in ESCs, Dicer acts as a repressor of antiviral responses and plays a key role in the maintenance of proliferation, viability, and pluripotency of ESCs.

Pluripotent stem cells are insensitive to the cytotoxicity of TNFα and IFNγ.

Recent studies have demonstrated that embryonic stem cells (ESCs) have an underdeveloped innate immune system, but the biological implications of this finding are poorly understood. In this study, we compared the responses of mouse ESCs (mESCs) and mESC differentiated fibroblasts (mESC-FBs) to tumor necrosis factor α (TNFα) and interferons (IFNs). Our data revealed that TNFα, IFNα, IFNß, or IFNγ alone do not cause apparent effects on mESCs and mESC-FBs, but the combination of TNFα and IFNγ (TNFα/IFNγ) showed toxicity to mESC-FBs as indicated by cell cycle inhibition and reduced cell viability, correlating with the expression of inducible nitric oxide synthase (iNOS). However, none of these effects were observed in mESCs that were treated with TNFα/IFNγ. Furthermore, mESC-FBs, but not mESCs, are vulnerable to cytotoxicity resulting from lipopolysaccharide (LPS)-activated macrophages. The insensitivity of mESCs to cytotoxicity in all cases is correlated with their lack of responses to TNFα and IFNγ. Similar to mESCs, human ESCs (hESCs) and iPSCs (hiPSCs) do not respond to TNFα and are not susceptible to the cytotoxicity of TNFα, IFNß, or IFNγ alone or in combination that significantly affects human foreskin fibroblast (hFBs) and Hela cells. However, unlike mESCs, hESCs and hiPSCs can respond to IFNγ, but this does not cause significant cytotoxicity in hESCs and hiPSCs. Our findings in both mouse and human PSCs together support the hypothesis that attenuated innate immune responses could be a protective mechanism that limits immunologic cytotoxicity resulting from inflammatory and immune responses.

Dietary iron deficiency impaired intestinal immune function of on-growing grass carp under the infection of Aeromonas hydrophila: Regulation of NF-κB and TOR signaling.

Iron is an important mineral element for fish. In this study, we investigated the influences of dietary iron deficiency on intestinal immune function as well as underlying signaling of on-growing grass carp (Ctenopharyngodon idella). Fish were fed with six graded level of dietary iron for sixty days, and a fourteen days' challenge test under infection of Aeromonas hydrophila thereafter. Results showed that compared with optimal iron level, iron deficiency increased enteritis morbidity, decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) concentrations and down-regulated mRNA levels of hepcidin, liver expressed antimicrobial peptide 2A (LEAP-2A), LEAP-2B, Mucin2, ß-defensin-1, anti-inflammatory cytokines transforming growth factor ß1 (TGF-ß1), TGF-ß2, interleukin 4/13A (IL-4/13A), IL-4/13B, IL-10, IL-11 and IL-15, inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1), whereas up-regulated mRNA levels of pro-inflammatory cytokines IL-1ß, interferon γ2 (IFN-γ2), IL-8, IL-12p35, IL-12p40 and IL-17D, nuclear factor kappa B (NF-κB) p65, IκB kinases α (IKKα), IKKß and eIF4E-binding protein (4E-BP) in intestine of on-growing grass carp, indicating that iron deficiency impaired intestinal immune function of fish under infection of A. hydrophila. Besides, iron excess also increased enteritis morbidity and impaired immune function of fish under infection of A. hydrophila. In addition, the effect of ferrous fumarate on intestinal immune function of on-growing grass carp is more efficient than ferrous sulfate. Finally, based on ability against enteritis, LZ activities in mid intestine and distal intestine, we recommended adding 83.37, 86.71 and 85.39 mg iron/kg into diet, respectively.

Role of endoplasmic reticulum stress and oxidative stress in vinyl chloride-induced hepatic steatosis in mice.

Vinyl chloride (VC) is a common industrial organochlorine, shown to cause hepatic angiosarcoma and hepatic steatosis. However, the role of endoplasmic reticulum stress (ERS) and oxidative stress (OS) in hepatic steatosis after subchronic exposure to VC in mice, is unclear. Based on body weight, forty healthy SPF male C57BL/6 J mice were randomly divided into a control group and three VC exposure groups (57.3, 286.7, and 1433.6 ppm) (n = 10 each). VC was administered by static inhalation in a 50 L sealed plexiglass inhalation chamber for 2 h per day, five days per week for 16 weeks. Serum and liver tissues were analyzed for liver enzymes and lipids. Hepatic cytochrome P450 2E1 (CYP2E1) and OS related indicators malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were measured. The mRNA expressions of ERS downstream genes, including glycoregulatory protein-78 (GRP-78), sterol regulatory element binding protein-1 (SREBP-1), Acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS) were detected by real-time PCR (RT-PCR) and their protein levels examined by western blotting. The CYP2E1 levels increased after VC administration in a dose-dependent manner. MDA levels increased (P < .05) and SOD and GSH levels decreased (P < .05) in the liver of each group with the increase in the dose of VC. ERS and expressions of downstream genes (GRP-78, SREBP-1, ACC, and FAS) were enhanced after VC administration. These results suggested that OS and ERS could be induced by VC, which may lead to an increase in fatty acid synthesis in the liver, further aggravating hepatic steatosis.

The underdeveloped innate immunity in embryonic stem cells: The molecular basis and biological perspectives from early embryogenesis.

Embryonic stem cells (ESCs) have been intensively studied as a promising cell source for regenerative medicine. The rapid advancements in the field have not only proven the feasibility of ESC-based cell therapy, but also led to a better understanding of pluripotent stem cells (PSCs) as a unique cell population at an early stage of embryogenesis. Recent studies have revealed that both human and mouse ESCs have attenuated innate immune responses to infectious agents and inflammatory cytokines. These findings raise interesting questions about the rationale for ESCs, the PSCs experimentally derived from preimplantation stage embryos, to not have an innate defense mechanism that has been adapted so well in somatic cells. All somatic cells have innate immune systems that can be activated by pathogen-associated molecular patterns (PAMPs) or cellular damage-associated molecular patterns (DAMPs), leading to production of cytokines. The underdeveloped innate immunity represents a unique property of PSCs that may have important implications. This review discusses the immunological properties of PSCs, the molecular basis underlying their diminished innate immune responses, and the hypothesis that the attenuated innate immune responses could be an adaptive mechanism that allows PSCs to avoid cytotoxicity associated with inflammation and immune responses during early embryogenesis.

Characterization of embryonic stem cell-differentiated fibroblasts as mesenchymal stem cells with robust expansion capacity and attenuated innate immunity.

BACKGROUND: Mesenchymal stem cells (MSCs) isolated from adult tissues (Ad-MSCs) have shown great promise for use in regenerative medicine. However, their poor in vitro expansion capacity and tissue scarcity have been major limitations. In this study, we demonstrate that mouse embryonic stem cells (mESCs) can differentiate into cells with MSC properties. METHODS: Using previously established methods that characterize Ad-MSCs, we analyzed mESC-differentiated fibroblasts (mESC-FBs), including plastic adherence, clonogenic growth, MSC marker expression, tri-lineage differentiation potential, and the capacity to express immunomodulators. RESULTS: Although previously characterized as mESC-differentiated fibroblasts (mESC-FBs), these cells exhibit major properties of Ad-MSCs. However, mESC-FBs also display unique features inherited from ESCs, including robust expansion capacity, senescence resistance, and attenuated innate immunity. In particular, mESC-FBs are insensitive to bacterial endotoxin (lipopolysaccharide, LPS) and do not express LPS-induced inflammatory molecules, in contrast to bone marrow (BM)-MSCs. We further demonstrate that mESC-FBs are resistant to the cytotoxicity associated with inflammatory cytokines, bacterial endotoxins (LPS and heat-killed bacteria), and macrophage-mediated inflammation. CONCLUSIONS: While it remains to be determined how the unique properties of mESC-FBs will affect their immunoregulatory activity under an in vivo condition, our findings demonstrate that ESCs could be used as an alternative source to generate a new class of ESC-MSCs with unique features potentially useful in regenerative medicine.

Congenital Zika Virus Infection in Immunocompetent Mice Causes Postnatal Growth Impediment and Neurobehavioral Deficits.

A small percentage of babies born to Zika virus (ZIKV)-infected mothers manifest severe defects at birth, including microcephaly. Among those who appeared healthy at birth, there are increasing reports of postnatal growth or developmental defects. However, the impact of congenital ZIKV infection in postnatal development is poorly understood. Here, we report that a mild congenital ZIKV-infection in pups born to immunocompetent pregnant mice did not display apparent defects at birth, but manifested postnatal growth impediments and neurobehavioral deficits, which include reduced locomotor and cognitive deficits that persisted into adulthood. We found that the brains of these pups were smaller, had a thinner cortical layer 1, displayed increased astrogliosis, decreased expression of microcephaly- and neuron development- related genes, and increased pathology as compared to mock-infected controls. In summary, our results showed that even a mild congenital ZIKV infection in immunocompetent mice could lead to postnatal deficits, providing definitive experimental evidence for a necessity to closely monitor postnatal growth and development of presumably healthy human infants, whose mothers were exposed to ZIKV infection during pregnancy.

The impaired immune function and structural integrity by dietary iron deficiency or excess in gill of fish after infection with Flavobacterium columnare: Regulation of NF-κB, TOR, JNK, p38MAPK, Nrf2 and MLCK signalling.

The aim of this study was to investigate the effects and potential mechanisms of dietary iron on immune function and structural integrity in gill of young grass carp (Ctenopharyngodon idella). A total of 630 grass carp (242.32 ±â€¯0.58 g) were fed diets containing graded levels of iron at 12.15 (basal diet), 35.38, 63.47, 86.43, 111.09, 136.37 and 73.50 mg/kg for 60 days. Subsequently, a challenge test was conducted by infection with Flavobacterium columnare to investigate the effects of dietary iron on gill immune function and structural integrity in young grass carp. First, the results indicated that compared with the optimal iron level, iron deficiency decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents, and down-regulated the mRNA levels of antibacterial peptides, anti-inflammatory cytokines (except IL-4/13B), inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1). In contrast, iron deficiency up-regulated the mRNA levels of pro-inflammatory cytokines (except IL-6 and IFN-γ2), nuclear factor κB p65 (NF-κBp65), IκB kinases α (IKK), IKKß, IKKγ, eIF4E-binding protein 1 (4E-BP1) and 4E-BP2 in gill of young grass carp, indicating that iron deficiency could impair immune function in fish gill. Second, iron deficiency down-regulated the mRNA levels of inhibitor of apoptosis protein (IAP) and myeloid cell leukemia 1 (Mcl-1), decreased activities and mRNA levels of antioxidant enzymes, down-regulated the mRNA levels of NF-E2-related factor 2 (Nrf2) and tight junction proteins (except claudin-12 and -15), and simultaneously increased malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) contents. Iron deficiency also up-regulated mRNA levels of cysteinyl aspartic acid-protease (caspase) -2, -7, -8, -9, Fas ligand (FasL), apoptotic protease activating factor-1 (Apaf-1), B-cell-lymphoma-2 associated X protein (Bax), p38 mitogen-activated protein kinase (p38MAPK), Kelch-like ECH-associating protein (Keap) 1a, Keap1b, claudin-12, -15 and MLCK, indicating that iron deficiency could disturb the structural integrity of gill in fish. Third, iron excess impaired immune function and structural integrity in gill of young grass carp. Forth, there was a better effect of ferrous fumarate than ferrous sulfate in young grass carp. Finally, the iron requirements based on ability against gill rot, ACP activity and MDA content in gill of young grass carp were estimated to be 76.52, 80.43 and 83.17 mg/kg, respectively.

The decreased growth performance and impaired immune function and structural integrity by dietary iron deficiency or excess are associated with TOR, NF-κB, p38MAPK, Nrf2 and MLCK signaling in head kidney, spleen and skin of grass carp (Ctenopharyngodon idella).

This study was conducted to investigate the effects of dietary iron on the growth, and immune function and structural integrity in head kidney, spleen and skin as well as the underlying signaling of young grass carp (Ctenopharyngodon idella). Total 630 grass carp (242.32 ± 0.58 g) were fed diets containing graded levels of iron at 12.15 (basal diet), 35.38, 63.47, 86.43, 111.09, 136.37 mg/kg (diets 2-6 were added with ferrous fumarate) and 73.50 mg/kg (diet 7 was added with ferrous sulfate) diet for 60 days. Then, a challenge test was conducted by infection of Aeromonas hydrophila for 14 days. The results firstly showed that compared with optimal iron level, iron deficiency decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents and down-regulated the mRNA levels of antibacterial peptides, anti-inflammatory cytokines, inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1), whereas up-regulated the mRNA levels of pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) p65, IκB kinases ß (IKKß) and eIF4E-binding protein (4E-BP) in head kidney and spleen of young grass carp (P < 0.05), indicating that iron deficiency impaired immune function in head kidney and spleen of fish. Secondly, iron deficiency down-regulated the mRNA levels of B-cell lymphoma-2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), and inhibitor of apoptosis protein (IAP), and decreased activities and mRNA levels of antioxidant enzymes, down-regulated the mRNA levels of NF-E2-related factor 2 (Nrf2) and tight junction complexes, and up-regulated mRNA levels of cysteinyl aspartic acid-protease (caspase) -2, -3, -7, -8, -9, apoptotic protease activating factor-1 (Apaf-1), Bcl-2 associated X protein (Bax), Fas ligand (FasL), p38 mitogen-activated protein kinase (p38MAPK), Kelch-like ECH-associating protein (Keap) 1a, Keap1b, claudin-12 and myosin light chain kinase (MLCK), and increased malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) contents in head kidney and spleen of young grass carp (P < 0.05), indicating that iron deficiency impaired structural integrity in head kidney and spleen of fish. Thirdly, iron deficiency increased skin hemorrhage and lesion morbidity, and impaired immune function and structural integrity in skin of fish. Fourthly, iron excess decreased growth and impaired the immune function and structural integrity in head kidney, spleen and skin of fish. Besides, in young grass carp, based on PWG and ability against skin hemorrhage and lesion, the efficacy of ferrous fumarate relative to ferrous sulfate was 140.32% and 126.48%, respectively, and the iron requirements based on PWG, ability against skin hemorrhage and lesion, ACP activities and MDA contents in head kidney and spleen were estimated to be 75.65, 87.03, 79.74, 78.93, 83.17 and 82.14 mg/kg diet (based on ferrous fumarate), respectively.

The Molecular Basis for the Lack of Inflammatory Responses in Mouse Embryonic Stem Cells and Their Differentiated Cells.

We reported previously that mouse embryonic stem cells do not have a functional IFN-based antiviral mechanism. The current study extends our investigation to the inflammatory response in mouse embryonic stem cells and mouse embryonic stem cell-differentiated cells. We demonstrate that LPS, TNF-α, and viral infection, all of which induce robust inflammatory responses in naturally differentiated cells, failed to activate NF-κB, the key transcription factor that mediates inflammatory responses, and were unable to induce the expression of inflammatory genes in mouse embryonic stem cells. Similar results were obtained in human embryonic stem cells. In addition to the inactive state of NF-κB, the deficiency in the inflammatory response in mouse embryonic stem cells is also attributed to the lack of functional receptors for LPS and TNF-α. In vitro differentiation can trigger the development of the inflammatory response mechanism, as indicated by the transition of NF-κB from its inactive to active state. However, a limited response to TNF-α and viral infection, but not to LPS, was observed in mouse embryonic stem cell-differentiated fibroblasts. We conclude that the inflammatory response mechanism is not active in mouse embryonic stem cells, and in vitro differentiation promotes only partial development of this mechanism. Together with our previous studies, the findings described in this article demonstrate that embryonic stem cells are fundamentally different from differentiated somatic cells in their innate immunity, which may have important implications in developmental biology, immunology, and embryonic stem cell-based regenerative medicine.

Utilization of different anti-viral mechanisms by mammalian embryonic stem cells and differentiated cells.

Embryonic stem cells (ESCs) have received tremendous attention because of their potential applications in regenerative medicine. Over the past two decades, intensive research has not only led to the generation of various types of cells from ESCs that can be potentially used for the treatment of human diseases but also led to the formation of new concepts and breakthroughs that have significantly impacted our understanding of basic cell biology and developmental biology. Recent studies have revealed that ESCs and other types of pluripotent cells do not have a functional interferon (IFN)-based anti-viral mechanism, challenging the idea that the IFN system is developed as the central component of anti-viral innate immunity in all types of cells in vertebrates. This finding also provided important insight into a question that has been uncertain for a long time: whether or not the RNA interference (RNAi) anti-viral mechanism operates in mammalian cells. An emerging paradigm is that mammals may have adapted distinct anti-viral mechanisms at different stages of organismal development; the IFN-based system is mainly used by differentiated somatic cells, while the RNAi anti-viral mechanism may be used in ESCs. This paper discusses the molecular basis and biological implications for mammals to have different anti-viral mechanisms during development.

Development of Antiviral Innate Immunity During In Vitro Differentiation of Mouse Embryonic Stem Cells.

The innate immunity of embryonic stem cells (ESCs) has recently emerged as an important issue in ESC biology and in ESC-based regenerative medicine. We have recently reported that mouse ESCs (mESCs) do not have a functional type I interferon (IFN)-based antiviral innate immunity. They are deficient in expressing IFN in response to viral infection and have limited ability to respond to IFN. Using fibroblasts (FBs) as a cell model, the current study investigated the development of antiviral mechanisms during in vitro differentiation of mESCs. We demonstrate that mESC-differentiated FBs (mESC-FBs) share extensive similarities with naturally differentiated FBs in morphology, marker expression, and growth pattern, but their development of antiviral mechanisms lags behind. Nonetheless, the antiviral mechanisms are inducible during mESC differentiation as demonstrated by the transition of nuclear factor kappa B (NFκB), a key transcription factor for IFN expression, from its inactive state in mESCs to its active state in mESC-FBs and by increased responses of mESC-FBs to viral stimuli and IFN during their continued in vitro propagation. Together with our previously published study, the current data provide important insights into molecular basis for the deficiency of IFN expression in mESCs and the development of antiviral innate immunity during mESC differentiation.

Attenuated Innate Immunity in Embryonic Stem Cells and Its Implications in Developmental Biology and Regenerative Medicine.

Embryonic stem cells (ESCs) represent a promising cell source for regenerative medicine. Intensive research over the past 2 decades has led to the feasibility of using ESC-differentiated cells (ESC-DCs) in regenerative medicine. However, increasing evidence indicates that ESC-DCs generated by current differentiation methods may not have equivalent cellular functions to their in vivo counterparts. Recent studies have revealed that both human and mouse ESCs as well as some types of ESC-DCs lack or have attenuated innate immune responses to a wide range of infectious agents. These findings raise important concerns for their therapeutic applications since ESC-DCs, when implanted to a wound site of a patient, where they would likely be exposed to pathogens and inflammatory cytokines. Understanding whether an attenuated immune response is beneficial or harmful to the interaction between host and grafted cells becomes an important issue for ESC-based therapy. A substantial amount of recent evidence has demonstrated that the lack of innate antiviral responses is a common feature to ESCs and other types of pluripotent cells. This has led to the hypothesis that mammals may have adapted different antiviral mechanisms at different stages of organismal development. The underdeveloped innate immunity represents a unique and uncharacterized property of ESCs that may have important implications in developmental biology, immunology, and in regenerative medicine.

Antiviral responses in mouse embryonic stem cells: differential development of cellular mechanisms in type I interferon production and response.

We have recently reported that mouse embryonic stem cells (mESCs) are deficient in expressing type I interferons (IFNs) in response to viral infection and synthetic viral RNA analogs (Wang, R., Wang, J., Paul, A. M., Acharya, D., Bai, F., Huang, F., and Guo, Y. L. (2013) J. Biol. Chem. 288, 15926-15936). Here, we report that mESCs are able to respond to type I IFNs, express IFN-stimulated genes, and mediate the antiviral effect of type I IFNs against La Crosse virus and chikungunya virus. The major signaling components in the IFN pathway are expressed in mESCs. Therefore, the basic molecular mechanisms that mediate the effects of type I IFNs are functional in mESCs; however, these mechanisms may not yet be fully developed as mESCs express lower levels of IFN-stimulated genes and display weaker antiviral activity in response to type I IFNs when compared with fibroblasts. Further analysis demonstrated that type I IFNs do not affect the stem cell state of mESCs. We conclude that mESCs are deficient in type I IFN expression, but they can respond to and mediate the cellular effects of type I IFNs. These findings represent unique and uncharacterized properties of mESCs and are important for understanding innate immunity development and ESC physiology.

Mouse embryonic stem cells have underdeveloped antiviral mechanisms that can be exploited for the development of mRNA-mediated gene expression strategy.

We have recently reported that mouse embryonic stem cells (mESCs) are deficient in expressing type I interferons (IFN) when exposed to viral infection and double-stranded RNA. In this study, we extended our investigation and demonstrated that single-stranded RNA and protein-encoding mRNA can induce strong IFN expression and cytotoxicity in fibroblasts and epithelial cells, but none of the effects associated with these antiviral responses were observed in mESCs. Our results provided additional data to support the conclusion that mESCs are intrinsically deficient in antiviral responses. While our findings represent a novel feature of mESCs that in itself is important for understanding innate immunity development, we exploited this property to develop a novel mRNA-mediated gene expression cell model. Direct introduction of synthetic mRNA to express desired genes has been shown as an effective alternative to DNA/viral vector-based gene expression. However, a major biological challenge is that a synthetic mRNA is detected as a viral RNA analog by the host cell, resulting in a series of adverse effects associated with antiviral responses. We demonstrate that the lack of antiviral responses in mESCs effectively avoids this problem. mESCs can tolerate repeated transfection and effectively express proteins from their synthetic mRNA with expected biological functions, as demonstrated by the expression of green fluorescent protein and the transcription factor Etv2. Therefore, mRNA-based gene expression could be developed into a novel ESC differentiation strategy that avoids safety concerns associated with viral/DNA-based vectors in regenerative medicine.