Innate Immunity in Human Embryonic Stem Cells: Comparison with Adult Human Endothelial Cells

University of Tor Vergata, Italy
PLoS ONE (Impact Factor: 3.23). 05/2010; 5(5):e10501. DOI: 10.1371/journal.pone.0010501
Source: PubMed


Treatment of human disease with human embryonic stem cell (hESC)-derived cells is now close to reality, but little is known of their responses to physiological and pathological insult. The ability of cells to respond via activation of Toll like receptors (TLR) is critical in innate immune sensing in most tissues, but also extends to more general danger sensing, e.g. of oxidative stress, in cardiomyocytes. We used biomarker release and gene-array analysis to compare responses in hESC before and after differentiation, and to those in primary human endothelial cells. The presence of cardiomyocytes and endothelial cells was confirmed in differentiated cultures by immunostaining, FACS-sorting and, for cardiomyocytes, beating activity. Undifferentiated hESC did not respond with CXCL8 release to Gram positive or Gram negative bacteria, or a range of PAMPs (pathogen associated molecular patterns) for TLRs 1-9 (apart from flagellin, an activator of TLR5). Surprisingly, lack of TLR-dependent responses was maintained over 4 months of differentiation of hESC, in cultures which included cardiomyocytes and endothelial cells. In contrast, primary cultures of human aortic endothelial cells (HAEC) demonstrated responses to a broad range of PAMPs. Expression of downstream TLR signalling pathways was demonstrated in hESC, and IL-1beta, TNFalpha and INFgamma, which bypass the TLRs, stimulated CXCL8 release. NFkappaB pathway expression was also present in hESC and NFkappaB was able to translocate to the nucleus. Low expression levels of TLRs were detected in hESC, especially TLRs 1 and 4, explaining the lack of response of hESC to the main TLR signals. TLR5 levels were similar between differentiated hESC and HAEC, and siRNA knockdown of TLR5 abolished the response to flagellin. These findings have potential implications for survival and function of grafted hESC-derived cells.

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    • "We have previously shown that endothelial cells derived from human embryonic stem cells (hESC-EC) express an immature immune phenotype, with no discernible TLR4 function [11]. We have speculated that this may provide an advantage since TLR4 on endothelial cells is directly linked to atherosclerosis [10], [11]. However, lack of TLR4 could result in endothelial cells not being able to sense pathogens, and render tissue/organs immune-suppressed and thereby susceptible to infection with Gram-negative bacteria. "
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    ABSTRACT: Human embryonic stem cell-derived endothelial cells (hESC-EC), as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR) Toll-like receptor (TLR)-4 and nucleotide-binding oligomerisation domain-containing protein (NOD)-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC). HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC), and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.
    PLoS ONE 04/2014; 9(4):e91119. DOI:10.1371/journal.pone.0091119 · 3.23 Impact Factor
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    • "Although a few genes that are part of the type I interferon response were upregulated following viral infection, there was an obvious lack in the expression of genes that are classically involved in innate immune responses to viral infection, such as nuclear factor-κB, type I interferons, interleukins and interferon regulatory factors (Geiss et al., 2001; Kamakura et al., 2008). This result was not unexpected, as significant upregulation of genes involved in the immune response were also not induced when hES or mES cells were infected with bacteria (Földes et al., 2010; Yu et al., 2009). This may be because transcription in ES cells is tightly regulated so that they remain pluripotent, and such regulation also includes interferon-stimulated genes (Szutorisz et al., 2006; Yu et al., 2009). "
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    ABSTRACT: Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA 'off-target' effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host-pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host-virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host-virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence.
    Journal of General Virology 07/2012; 93(Pt 10):2118-30. DOI:10.1099/vir.0.043406-0 · 3.18 Impact Factor
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    • "The cardiovascular system is exposed to invading pathogens and pathogen-derived molecules at both systemic and local levels (involving blood and blood vessels). It is recognized that endothelial cells are the first tissue to sense and respond to pathogens (or PAMPs) via the activation of cytokines, chemokines, and dilator hormones [79], facilitating transfer and migration of leukocytes at the site of injury and increased blood flow to promote resolution [80]. In addition to the endothelium, underlying vascular smooth muscle cells (VSMCs) can also sense PAMPs via TLRs and NLRs which result in the activation of vasoactive hormones and increased flow [81]. "
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    ABSTRACT: NOD-like receptors (NLRs) constitute a recently identified family of macromolecules that participate in regulation of innate immune responses. To date, 23 members of the NLR family are identified in humans. Diverse NLRs are stimulated by a broad range of pathogen- or danger-associated molecular patterns, and collectively function as intracellular pattern recognition receptors (PRRs). The most studied inflammasomes are NLRP1 and NLRP3 that process inactive pro-caspase-1 to its active form, allowing the cleavage and subsequent activation of pro-IL-1β and pro-IL-18, and initiation of inflammatory responses. Three models, based upon extracellular ATP/K(+) flux, lysosomal release of cathepsin, and reactive oxygen species, have been proposed to be involved in signaling activation of NLRs and downstream events. In this review, I discuss the current state of knowledge related to the roles of NLRs and inflammasomes in the development of cardiovascular diseases.
    American Journal of Cardiovascular Disease 01/2011; 1(3):244-54.
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