scholarly journals Rotavirus Degrades Multiple Interferon (IFN) Type Receptors To Inhibit IFN Signaling and Protects against Mortality from Endotoxin in Suckling Mice

2017 ◽  
Vol 92 (1) ◽  
Author(s):  
Adrish Sen ◽  
Ayushi Sharma ◽  
Harry B. Greenberg
Keyword(s):  
Intestinal Epithelial Cells ◽  
Degradation Pathway ◽  
Type I ◽  
Intestinal Epithelial ◽  
Genetic Ablation ◽  
Suckling Mice ◽  
Infected Cells ◽  
New Research

ABSTRACTSTAT1 phosphorylation in response to exogenous interferon (IFN) administration can be inhibited by rotaviral replication bothin vitroandin vivo. In addition many rotavirus strains are resistant to the actions of different IFN types. The regulation by rotaviruses (RVs) of antiviral pathways mediated by multiple IFN types is not well understood. In this study, we find that during infectionin vitroandin vivo, RVs significantly deplete IFN type I, II, and III receptors (IFNRs). Regulation of IFNRs occurred exclusively within RV-infected cells and could be abrogated by inhibiting the lysosomal-endosomal degradation pathway.In vitro, IFNR degradation was conserved across multiple RV strains that differ in their modes of regulating IFN induction. In suckling mice, exogenously administered type I, II, or III IFN induced phosphorylation of STAT1-Y701 within intestinal epithelial cells (IECs) of suckling mice. Murine EW strain RV infection transiently activated intestinal STAT1 at 1 day postinfection (dpi) but not subsequently at 2 to 3 dpi. In response to injection of purified IFN-α/β or -λ, IECs in EW-infected mice exhibited impaired STAT1-Y701 phosphorylation, correlating with depletion of different intestinal IFNRs and impaired IFN-mediated transcription. The ability of EW murine RV to inhibit multiple IFN types led us to test protection of suckling mice from endotoxin-mediated shock, an outcome that is dependent on the host IFN response. Compared to mortality in controls, mice infected with EW murine RV were substantially protected against mortality following parenteral endotoxin administration. These studies identify a novel mechanism of IFN subversion by RV and reveal an unexpected protective effect of RV infection on endotoxin-mediated shock in suckling mice.IMPORTANCEAntiviral functions of types I, II, and III IFNs are mediated by receptor-dependent activation of STAT1. Here, we find that RV degrades the types I, II, and III IFN receptors (IFNRs)in vitro. In a suckling mouse model, RV effectively blocked STAT1 activation and transcription following injection of different purified IFNs. This correlated with significantly decreased protein expression of intestinal types I and II IFNRs. Recent studies demonstrate that in mice lipopolysaccharide (LPS)-induced lethality is prevented by genetic ablation of IFN signaling genes such as IFNAR1 and STAT1. When suckling mice were infected with RV, they were substantially protected from lethal exposure to endotoxin. These findings provide novel insights into the mechanisms underlying rotavirus regulation of different interferons and are likely to stimulate new research into both rotavirus pathogenesis and endotoxemia.

Progastrin1–80stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites

2003 ◽  
Vol 284 (2) ◽  
pp. G328-G339 ◽  
Author(s):  
P. Singh ◽  
X. Lu ◽  
S. Cobb ◽  
B. T. Miller ◽  
N. Tarasova ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Binding Sites ◽  
Intestinal Epithelial Cells ◽  
Full Length ◽  
Intestinal Epithelial ◽  
Growth Effects ◽  
High Affinity ◽  
Significant Difference

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhPG1–80) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1–1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CCK1and CCK2receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK1-R and CCK2-R on IEC cells. High-affinity ( Kd= 0.5–1.0 nM) binding sites for125I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG ≥ COOH-terminally extended G17 ≥ G-Gly > G17 > *CCK-8 (* significant difference; P< 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.

scholarly journals HIV-Infected Macrophages Are Infected and Killed by the Interferon-Sensitive Rhabdovirus MG1

2021 ◽  
Vol 95 (9) ◽  
Author(s):  
Teslin S. Sandstrom ◽  
Nischal Ranganath ◽  
Stephanie C. Burke Schinkel ◽  
Syim Salahuddin ◽  
Oussama Meziane ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Oncolytic Viruses ◽  
Type I Interferons ◽  
Viral Reservoir ◽  
Type I ◽  
Antiviral Signaling ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT The use of unique cell surface markers to target and eradicate HIV-infected cells has been a longstanding objective of HIV-1 cure research. This approach, however, overlooks the possibility that intracellular changes present within HIV-infected cells may serve as valuable therapeutic targets. For example, the identification of dysregulated antiviral signaling in cancer has led to the characterization of oncolytic viruses capable of preferentially killing cancer cells. Since impairment of cellular antiviral machinery has been proposed as a mechanism by which HIV-1 evades immune clearance, we hypothesized that HIV-infected macrophages (an important viral reservoir in vivo) would be preferentially killed by the interferon-sensitive oncolytic Maraba virus MG1. We first showed that HIV-infected monocyte-derived macrophages (MDM) were more susceptible to MG1 infection and killing than HIV-uninfected cells. As MG1 is highly sensitive to type I interferons (IFN-I), we then investigated whether we could identify IFN-I signaling differences between HIV-infected and uninfected MDM and found evidence of impaired IFN-α responsiveness within HIV-infected cells. Finally, to assess whether MG1 could target a relevant, primary cell reservoir of HIV-1, we investigated its effects in alveolar macrophages (AM) obtained from effectively treated individuals living with HIV-1. As observed with in vitro-infected MDM, we found that HIV-infected AM were preferentially eliminated by MG1. In summary, the oncolytic rhabdovirus MG1 appears to preferentially target and kill HIV-infected cells via impairment of antiviral signaling pathways and may therefore provide a novel approach to an HIV-1 cure. IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) remains a treatable, but incurable, viral infection. The establishment of viral reservoirs containing latently infected cells remains the main obstacle in the search for a cure. Cure research has also focused on only one cellular target of HIV-1 (the CD4+ T cell) while largely overlooking others (such as macrophages) that contribute to HIV-1 persistence. In this study, we address these challenges by describing a potential strategy for the eradication of HIV-infected macrophages. Specifically, we show that an engineered rhabdovirus—initially developed as a cancer therapy—is capable of preferential infection and killing of HIV-infected macrophages, possibly via the same altered antiviral signaling seen in cancer cells. As this rhabdovirus is currently being explored in phase I/II clinical trials, there is potential for this approach to be readily adapted for use within the HIV-1 cure field.

scholarly journals Transcriptional role of p53 in interferon-mediated antiviral immunity

2008 ◽  
Vol 205 (8) ◽  
pp. 1929-1938 ◽  
Author(s):  
César Muñoz-Fontela ◽  
Salvador Macip ◽  
Luis Martínez-Sobrido ◽  
Lauren Brown ◽  
Joseph Ashour ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Viral Replication ◽  
Viral Infections ◽  
Suppressor Gene ◽  
Central Component ◽  
Type I ◽  
Infected Cells

Tumor suppressor p53 is activated by several stimuli, including DNA damage and oncogenic stress. Previous studies (Takaoka, A., S. Hayakawa, H. Yanai, D. Stoiber, H. Negishi, H. Kikuchi, S. Sasaki, K. Imai, T. Shibue, K. Honda, and T. Taniguchi. 2003. Nature. 424:516–523) have shown that p53 is also induced in response to viral infections as a downstream transcriptional target of type I interferon (IFN) signaling. Moreover, many viruses, including SV40, human papillomavirus, Kaposi's sarcoma herpesvirus, adenoviruses, and even RNA viruses such as polioviruses, have evolved mechanisms designated to abrogate p53 responses. We describe a novel p53 function in the activation of the IFN pathway. We observed that infected mouse and human cells with functional p53 exhibited markedly decreased viral replication early after infection. This early inhibition of viral replication was mediated both in vitro and in vivo by a p53-dependent enhancement of IFN signaling, specifically the induction of genes containing IFN-stimulated response elements. Of note, p53 also contributed to an increase in IFN release from infected cells. We established that this p53-dependent enhancement of IFN signaling is dependent to a great extent on the ability of p53 to activate the transcription of IFN regulatory factor 9, a central component of the IFN-stimulated gene factor 3 complex. Our results demonstrate that p53 contributes to innate immunity by enhancing IFN-dependent antiviral activity independent of its functions as a proapoptotic and tumor suppressor gene.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

7 LACTOBACILLUS REUTERI SUPPRESSES PRO-INFLAMMATORY DRIVEN REACTIVE OXYGEN SPECIES IN VITRO IN HUMAN INTESTINAL EPITHELIAL CELLS AND IN VIVO IN A TNBS COLITIS MOUSE MODEL

2020 ◽  
Vol 26 (Supplement_1) ◽  
pp. S41-S41 ◽  
Author(s):  
Wenly Ruan ◽  
Melinda Engevik ◽  
Alexandra Chang-Graham ◽  
Joseph Hyser ◽  
James Versalovic
Keyword(s):  
Reactive Oxygen Species ◽  
Epithelial Cells ◽  
Lactobacillus Reuteri ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial Cells ◽  
Oxygen Species ◽  
Intestinal Epithelial ◽  
Colitis Model

Abstract Background Reactive oxygen species (ROS) play a role in maintaining intestinal epithelial homeostasis and are normally kept at low levels via antioxidant compounds. Dysregulation of ROS can lead to intestinal inflammation and contribute to inflammatory bowel disease (IBD). Select gut microbes possess the enzymatic machinery to produce antioxidants whereas others can dysregulate levels of ROS. Our model microbe, Lactobacillus reuteri (ATCC PTA 6475), has been demonstrated to reduce intestinal inflammation in mice models. It contains the genes encoding two distinct GshA-like glutamylcysteine ligases. We hypothesize that L. reuteri can secrete γ-glutamylcysteine to suppress ROS, minimize NFκB activation and regulate secretion of e pithelial cytokines. Methods & Results Conditioned media from L. reuteri was analyzed via mass spectrometry to confirm the presence of γ-glutamylcysteine. All cysteine containing products including γ-glutamylcysteine were fluorescently tagged in the conditioned media and then incubated with HT29 cell monolayers as well as human jejunal enteroid (HJE) monolayers. γ-glutamylcysteine was demonstrated to enter intestinal epithelial cells based on microscopy. Next, a Thioltracker assay was used to show increased intracellular glutathione levels by L. reuteri secreted γ-glutamylcysteine. HT29 cells and HJEs were then treated with IL-1β or hydrogen peroxide, and L. reuteri metabolites as well as γ-glutamylcysteine significantly suppressed pro-inflammatory cytokine driven ROS and IL-8 production. L. reuteri secreted products also reduced activity of NFκB as determined by a luciferase reporter assay. γ-glutamylcysteine deficient mutants were generated by targeted mutagenesis of GshA genes, and these mutant L. reuteri strains had a diminished ability to suppress IL-8 production and ROS. To further test the role of L. reuteri secreted γ-glutamylcysteine in vivo, a 2,4,6-Trinitrobenzenesulfonic acid (TNBS)- induced mouse colitis model was used. Adolescent mice were orogavaged with PBS, L. reuteri, L. reuteri GshA2 mutant, or γ-glutamylcysteine for a week after which TNBS was rectally administered to induce colitis. We demonstrate that L. reuteri and γ-glutamylcysteine can suppress histologic inflammation compared to PBS control and L. reuteri GshA2 mutant groups. Conclusions Together these data indicate that L. reuteri secretes γ-glutamylcysteine which can enter the intestinal epithelial cells and modulate epithelial cytokine production. It acts via suppression of ROS and NFκB which then decreases IL-8 production. We are able to demonstrate this in vitro in both HT 29 cells and HJEs. We now also demonstrate this in vivo in a mouse colitis model. These experiments highlight a prominent role for ROS intermediates in microbiome-mammalian cell signaling processes involved in immune responses and intestinal inflammation.

scholarly journals Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Gisselle N. Medina ◽  
Paul Azzinaro ◽  
Elizabeth Ramirez-Medina ◽  
Joseph Gutkoska ◽  
Ying Fang ◽  
...  
Keyword(s):  
Disease Virus ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Type I ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Infected Cells ◽  
Viral Attenuation

ABSTRACT Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) affects several pathways of the host innate immune response. Previous studies in bovine cells demonstrated that deletions (leaderless [LLV]) or point mutations in Lpro result in increased expression of interferon (IFN) and IFN-stimulated genes (ISGs), including, among others, the ubiquitin-like protein modifier ISG15 and the ubiquitin specific peptidase USP18. In addition to its conventional papain-like protease activity, Lpro acts as a deubiquitinase (DUB) and deISGylase. In this study, we identified a conserved residue in Lpro that is involved in its interaction with ISG15. Mutation W105A rendered Escherichia coli-expressed Lpro unable to cleave the synthetic substrate pro-ISG15 while preserving cellular eIF4G cleavage. Interestingly, mutant FMDV W105A was viable. Overexpression of ISG15 and the ISGylation machinery in porcine cells resulted in moderate inhibition of FMDV replication, along with a decrease of the overall state of ISGylation in wild-type (WT)-infected cells. In contrast, reduced deISGylation was observed upon infection with W105A and leaderless virus. Reduction in the levels of deubiquitination was also observed in cells infected with the FMDV LproW105A mutant. Surprisingly, similarly to WT, infection with W105A inhibited IFN/ISG expression despite displaying an attenuated phenotype in vivo in mice. Altogether, our studies indicate that abolishing/reducing the deISGylase/DUB activity of Lpro causes viral attenuation independently of its ability to block the expression of IFN and ISG mRNA. Furthermore, our studies highlight the potential of ISG15 to be developed as a novel biotherapeutic molecule against FMD. IMPORTANCE In this study, we identified an aromatic hydrophobic residue in foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) (W105) that is involved in the interaction with ISG15. Mutation in Lpro W105 (A12-LproW105A) resulted in reduced deISGylation in vitro and in porcine-infected cells. Impaired deISGylase activity correlated with viral attenuation in vitro and in vivo and did not affect the ability of Lpro to block expression of type I interferon (IFN) and other IFN-stimulated genes. Moreover, overexpression of ISG15 resulted in the reduction of FMDV viral titers. Thus, our study highlights the potential use of Lpro mutants with modified deISGylase activity for development of live attenuated vaccine candidates, and ISG15 as a novel biotherapeutic against FMD.

Flavonoids alter P-gp expression in intestinal epithelial cells in vitro and in vivo

2007 ◽  
Vol 51 (3) ◽  
pp. 293-300 ◽  
Author(s):  
Katrin Lohner ◽  
Kerstin Schnäbele ◽  
Hannelore Daniel ◽  
Doris Oesterle ◽  
Gerhard Rechkemmer ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial

scholarly journals A cytomegaloviral protein reveals a dual role for STAT2 in IFN-γ signaling and antiviral responses

2005 ◽  
Vol 201 (10) ◽  
pp. 1543-1553 ◽  
Author(s):  
Albert Zimmermann ◽  
Mirko Trilling ◽  
Markus Wagner ◽  
Manuel Wilborn ◽  
Ivan Bubic ◽  
...  
Keyword(s):  
Biological Significance ◽  
Receptor Expression ◽  
Type I ◽  
Antiviral Responses ◽  
Type I Ifns ◽  
New Strategy ◽  
Infected Cells ◽  
Ifn Γ

A mouse cytomegalovirus (MCMV) gene conferring interferon (IFN) resistance was identified. This gene, M27, encodes a 79-kD protein that selectively binds and down-regulates for signal transducer and activator of transcription (STAT)-2, but it has no effect on STAT1 activation and signaling. The absence of pM27 conferred MCMV susceptibility to type I IFNs (α/β), but it had a much more dramatic effect on type II IFNs (γ) in vitro and in vivo. A comparative analysis of M27+ and M27− MCMV revealed that the antiviral efficiency of IFN-γ was partially dependent on the synergistic action of type I IFNs that required STAT2. Moreover, STAT2 was directly activated by IFN-γ. This effect required IFN receptor expression and was independent of type I IFNs. IFN-γ induced increasing levels of tyrosine-phosphorylated STAT2 in M27− MCMV-infected cells that were essential for the antiviral potency of IFN-γ. pM27 represents a new strategy for simultaneous evasions from types I and II IFNs, and it documents an unknown biological significance for STAT2 in antiviral IFN-γ responses.

Some factors affecting the adherence of probiotic Propionibacterium acidipropionici CRL 1198 to intestinal epithelial cells

2002 ◽  
Vol 48 (5) ◽  
pp. 449-457 ◽  
Author(s):  
Gabriela Zárate ◽  
Vilma Morata De Ambrosini ◽  
Adriana Perez Chaia ◽  
Silvia González
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Divalent Cations ◽  
Metabolic Effects ◽  
Bacterial Cells ◽  
Intestinal Epithelial ◽  
Propionibacterium Acidipropionici ◽  
Sodium Metaperiodate

Adhesion to the intestinal mucosa is generally considered an important property of probiotic microorganisms and has been related to many of their health benefits. This study investigated some factors that could affect or be involved in the adherence of Propionibacterium acidipropionici CRL 1198, a dairy strain with suggested probiotic effects and high adherence in vitro and in vivo to intestinal epithelial cells. In vitro adhesion of propionibacteria was decreased by gastric digestion but not affected by bile and pancreatic enzymes. Adherence was also decreased by pretreatment of bacterial cells with protease, sodium metaperiodate, and trichloroacetic acid, revealing that different features of the cell surface, like protein factors, carbohydrates, and teichoic acids, are involved in the process. Adherence to intestinal epithelial cells was enhanced by calcium and was dependent on other divalent cations. Adhesion to intestinal mucus was also demonstrated. The results should explain the metabolic effects in the host previously obtained with this strain and support the potential of Propionibacterium for development of new probiotics.Key words: propionibacteria, adhesion, probiotics.

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