hTERT Extends the Life of Human Fibroblasts without Compromising Type I Interferon Signaling

Smith, Miles; Goddard, Erica T.; Lanfranca, Mirna Perusina; Davido, David J.

doi:10.1371/journal.pone.0058233

Cited by 19 publications

(19 citation statements)

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“…Since these cells merely have extended life via telomerase and are not transformed via mutagenesis, they represent a suitable model of the innate immune signaling that is often defective in cancerous cell lines (56). Importantly, type I IFN responses are fully functional in these cells (57). Furthermore, their prolonged life span renders them tractable to transgenic manipulations, such as gene knockout or protein overexpression, that rely on multiple passages in the presence of selecting agents.…”

Section: Discussionmentioning

confidence: 99%

Emerging Alphaviruses Are Sensitive to Cellular States Induced by a Novel Small-Molecule Agonist of the STING Pathway

Gall

Pryke

Abraham

et al. 2018

J Virol

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The type I interferon (IFN) system represents an essential innate immune response that renders cells resistant to virus growth via the molecular actions of IFN-induced effector proteins. IFN-mediated cellular states inhibit growth of numerous and diverse virus types, including those of known pathogenicity as well as potentially emerging agents. As such, targeted pharmacologic activation of the IFN response may represent a novel therapeutic strategy to prevent infection or spread of clinically impactful viruses. In light of this, we employed a high-throughput screen to identify small molecules capable of permeating the cell and of activating IFN-dependent signaling processes. Here we report the identification and characterization of -(methylcarbamoyl)-2-{[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-2-phenylacetamide (referred to as C11), a novel compound capable of inducing IFN secretion from human cells. Using reverse genetics-based loss-of-function assays, we show that C11 activates the type I IFN response in a manner that requires the adaptor protein STING but not the alternative adaptors MAVS and TRIF. Importantly, treatment of cells with C11 generated a cellular state that potently blocked replication of multiple emerging alphavirus types, including chikungunya, Ross River, Venezuelan equine encephalitis, Mayaro, and O'nyong-nyong viruses. The antiviral effects of C11 were subsequently abrogated in cells lacking STING or the type I IFN receptor, indicating that they are mediated, at least predominantly, by way of STING-mediated IFN secretion and subsequent autocrine/paracrine signaling. This work also allowed characterization of differential antiviral roles of innate immune signaling adaptors and IFN-mediated responses and identified MAVS as being crucial to cellular resistance to alphavirus infection. Due to the increase in emerging arthropod-borne viruses, such as chikungunya virus, that lack FDA-approved therapeutics and vaccines, it is important to better understand the signaling pathways that lead to clearance of virus. Here we show that C11 treatment makes human cells refractory to replication of a number of these viruses, which supports its value in increasing our understanding of the immune response and viral pathogenesis required to establish host infection. We also show that C11 depends on signaling through STING to produce antiviral type I interferon, which further supports its potential as a therapeutic drug or research tool.

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Section: Discussionmentioning

confidence: 99%

Emerging Alphaviruses Are Sensitive to Cellular States Induced by a Novel Small-Molecule Agonist of the STING Pathway

Gall

Pryke

Abraham

et al. 2018

J Virol

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“…Immortalized primary cells can provide an accurate model for studying virus–host interactions if they are derived in such a way that preserves the integrity of innate antiviral signaling. Different cell types have been immortalized by exogenous hTERT expression, and hTERT-immortalized fibroblasts have been observed to have intact IFN-I and IFN-independent antiviral responses [75]. Thus, with sufficient care and characterization, relevant studies can be performed to elucidate fundamental aspects of virus–host interactions.…”

Section: Discussionmentioning

confidence: 99%

“…Fibroblasts were the original cell type immortalized using hTERT and hTERT-immortalized fibroblasts closely resemble primary cells, have intact p53/pRb signaling, and have normal responses to serum starvation, contact inhibition, or DNA damage [73,74]. Importantly, hTERT immortalized fibroblasts can undergo greater than 100 population doublings under the right conditions and exogenous hTERT expression does not impact IFN-I signaling and permissivity to herpex simplex virus (HSV) or VSV [75]. However, the effects on antiviral signaling of hTERT-immortalization in other cell types or following continual passaging have not been directly investigated.…”

Section: Developing a Cellular Model To Study Virus–host Interactimentioning

confidence: 99%

The Importance of Physiologically Relevant Cell Lines for Studying Virus–Host Interactions

Hare

Collins

Cuddington

et al. 2016

Viruses

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Viruses interact intimately with the host cell at nearly every stage of replication, and the cell model that is chosen to study virus infection is critically important. Although primary cells reflect the phenotype of healthy cells in vivo better than cell lines, their limited lifespan makes experimental manipulation challenging. However, many tumor-derived and artificially immortalized cell lines have defects in induction of interferon-stimulated genes and other antiviral defenses. These defects can affect virus replication, especially when cells are infected at lower, more physiologically relevant, multiplicities of infection. Understanding the selective pressures and mechanisms underlying the loss of innate signaling pathways is helpful to choose immortalized cell lines without impaired antiviral defense. We describe the trials and tribulations we encountered while searching for an immortalized cell line with intact innate signaling, and how directed immortalization of primary cells avoids many of the pitfalls of spontaneous immortalization.

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“…Primary human foetal lung fibroblast (MRC5) cells were purchased from the European Collection of Authenticated Cell Cultures (ECACC; 05072101). MRC5t cells are immortalized MRC5 cells expressing the catalytic subunit of human telomerase (hTERT), and were generated as previously described [92]. MRC5 and MRC5t cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies; 41966) supplemented with 10 % foetal bovine serum (FBS; Life Technologies; 10270), 100 U/ml of penicillin and 100 μg/ml of streptomycin (P/S; Life Technologies; 15140-122), and 1× non-essential amino acids (NEAA; Life Technologies 11140-035).…”

Section: Methodsmentioning

confidence: 99%

Constitutive TRIM22 expression within the respiratory tract identifies tissue-specific and cell-type dependent intrinsic immune barriers to influenza A virus infection

Charman

McFarlane

Wojtus

et al. 2019

Preprint

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23 We hypothesized that increased expression of antiviral host factors at portals of viral entry 24 may protect exposed tissues from the constant threat of invading pathogens. Comparative 25 transcriptomic analysis identified the broad-acting restriction factor TRIM22 (TRIpartite 26 Motif 22) to be among the most abundantly expressed antiviral host factors in the lung, a 27 major portal of entry for many respiratory pathogens. This was surprising, as TRIM22 is 28 currently considered to be an interferon stimulated gene (ISG) product that confers protection 29 following the activation of pathogen-induced cytokine-mediated innate immune defences.30 Using human respiratory cell lines and the airways of rhesus macaques, we experimentally 31 confirmed high levels of constitutive TRIM22 expression in the lung. In contrast, TRIM22 32 expression in many widely used transformed cell lines could only be observed following 33 immune stimulation. Endogenous levels of TRIM22 in non-transformed cells were sufficient 34 to restrict human and avian influenza A virus (IAV) infection by inhibiting the onset of viral 35 transcription independently of cytokine-mediated innate immune defences. Thus, TRIM22 36 confers a pre-existing (intrinsic) tissue-specific immune barrier to IAV infection in the 37 respiratory tract. We investigated whether the constitutive expression of TRIM22 was a 38 characteristic shared by other ISGs in human lung tissue. Transcriptomic analysis identified a 39 large group of ISGs and IAV immuno-regulatory host factors that were similarly enriched in 40 the lung relative to other mucosal tissues, but whose expression was downregulated in 41 transformed cell-lines. We identify common networks of immune gene downregulation 42 which correlated with enhanced permissivity of transformed cells to initiate IAV replication.43 Our data highlight the importance of tissue-specific and cell-type dependent patterns of pre-44 existing immune gene expression in the intrinsic intracellular restriction of IAV; findings 45 highly relevant to the immune regulation of many clinically important respiratory pathogens. 47Author Summary 48 The respiratory tract is a major portal of virus entry for many clinically important viruses, 49 including seasonal and pandemic influenza A virus (IAV). We reasoned that cells within the 50 respiratory tract might differentially express antiviral host factors to protect against the 51 constant challenge of viral infection. We found the broad-acting antiviral protein TRIM22, 52 conventionally regarded as an interferon stimulated gene (ISG) product upregulated in 53 response to virus infection, to be constitutively expressed to high levels in the lung. We found 54 that constitutive expression of TRIM22 restricted the initiation of human and avian IAV 55 infection independently of cytokine-mediated innate immune defences. We identified pre-56 existing tissue-specific and cell-type dependent patterns of constitutive immune gene 57 expression that strongly correlated with enhanced resistance to IAV r...

show abstract

hTERT Extends the Life of Human Fibroblasts without Compromising Type I Interferon Signaling

Cited by 19 publications

References 100 publications

Emerging Alphaviruses Are Sensitive to Cellular States Induced by a Novel Small-Molecule Agonist of the STING Pathway

Emerging Alphaviruses Are Sensitive to Cellular States Induced by a Novel Small-Molecule Agonist of the STING Pathway

The Importance of Physiologically Relevant Cell Lines for Studying Virus–Host Interactions

Constitutive TRIM22 expression within the respiratory tract identifies tissue-specific and cell-type dependent intrinsic immune barriers to influenza A virus infection

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