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A TLR and non-TLR mediated innate response to lentiviruses restricts hepatocyte entry and can be ameliorated by pharmacological blockade.

ABSTRACT: Lentiviral vector (LV)-mediated gene transfer is a promising method of gene therapy. We previously reported that systemic injection of HIV-based LV triggers a transient inflammatory response. Here, we carried out studies to better characterize this response, and to develop a strategy to overcome the adverse effects of interferon (IFN) on LV-mediated gene transfer. We profiled gene expression in the liver after LV administration using deep-sequencing (RNA-seq), and identified several innate response pathways. We examined the response to LV in MyD88-TRIF knockout mice, which are incapable of toll-like receptor (TLR) signaling. Unexpectedly, the IFN response to LV was not reduced in the liver indicating that a non-TLR pathway can recognize LV in this organ. Indeed, blocking reverse transcription with azidothymidine (AZT) reduced the IFN response only in the liver, suggesting that proviral DNA can be a trigger. To block the inflammatory response, we pretreated mice with a short course of dexamethasone (Dex). At 4 hours post-treatment, all the IFN-induced genes were normalized. By blocking the inflammatory response, hepatocyte transduction was dramatically increased, which in turn doubled the level of human factor IX (FIX) produced by a hepatocyte-specific LV. Our studies uncover new insights into LV-induced immune responses in the liver, and provide a means to increase the safety and efficiency of LV-mediated gene transfer.

SUBMITTER: Agudo J

PROVIDER: S-EPMC3519984 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A TLR- and non-TLR-mediated innate response to lentiviruses restricts hepatocyte entry and can be ameliorated by pharmacological blockade

Project description:Lentiviral vector (LV)-mediated gene transfer is a promising method of gene therapy. We previously reported that systemic injection of LV triggers a transient inflammatory response. Here, we carried out studies to better characterize this response, and to develop a strategy to overcome the effects of interferon (IFN) on LV-mediated gene transfer. We profiled gene expression in the liver after LV administration using deep-sequencing, and identified several innate response pathways. We examined the response to LV in MyD88-TRIF knock-out mice, which are incapable of toll-like receptor (TLR) signaling. The IFN response to LV was not reduced in the liver, indicating that a non-TLR pathway can recognize LV in this tissue. Indeed, blocking reverse-transcription with AZT reduced the IFN response only in the liver, suggesting that proviral DNA can be a trigger. To block the inflammatory response, we pre-treated mice with a short-course of dexamethasone. At 4 hours post-treatment, all of the IFN-induced genes were normalized. By blocking the inflammatory response, hepatocyte transduction was dramatically increased, which doubled the level of human factor-IX produced by a hepatocyte-specific LV. Our studies uncover new insights into LV-induced immune responses in the liver, and provide a means to increase the safety and efficiency of LV-mediated gene transfer. mRNA profiles from livers of untreated and LV-treated mice were generated by deep-sequencing in Illumina HiSeq 2000.

2013-07-05 | E-GEOD-39129 | biostudies-arrayexpress

A TLR- and non-TLR-mediated innate response to lentiviruses restricts hepatocyte entry and can be ameliorated by pharmacological blockade

2013-07-05 | GSE39129 | GEO

Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis.

Project description:Despite the wide administration of several effective vaccines, rotavirus (RV) remains the single most important etiological agent of severe diarrhea in infants and young children worldwide, with an annual mortality of over 200,000 people. RV attachment and internalization into target cells is mediated by its outer capsid protein VP4. To better understand the molecular details of RV entry, we performed tandem affinity purification coupled with high-resolution mass spectrometry to map the host proteins that interact with VP4. We identified an actin-binding protein, drebrin (DBN1), that coprecipitates and colocalizes with VP4 during RV infection. Importantly, blocking DBN1 function by siRNA silencing, CRISPR knockout (KO), or chemical inhibition significantly increased host cell susceptibility to RV infection. Dbn1 KO mice exhibited higher incidence of diarrhea and more viral antigen shedding in their stool samples compared with the wild-type littermates. In addition, we found that uptake of other dynamin-dependent cargos, including transferrin, cholera toxin, and multiple viruses, was also enhanced in DBN1-deficient cells. Inhibition of cortactin or dynamin-2 abrogated the increased virus entry observed in DBN1-deficient cells, suggesting that DBN1 suppresses dynamin-mediated endocytosis via interaction with cortactin. Our study unveiled an unexpected role of DBN1 in restricting the entry of RV and other viruses into host cells and more broadly to function as a crucial negative regulator of diverse dynamin-dependent endocytic pathways.

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HSV ICP0 recruits USP7 to modulate TLR-mediated innate response.

Project description:Pattern recognition receptors represent the first line of defense against invading pathogens. Herpes simplex virus (HSV) encodes multiple ligands detected by these receptors, yet persists in the majority of infected individuals indicating a breakdown in host defense against the virus. Here we identify a novel mechanism through which HSV immediate-early protein ICP0 inhibits TLR-dependent inflammatory response by blocking NF-kappaB and JNK activation downstream of TLR signal activation. This process depends on ICP0-mediated translocation of USP7 (HAUSP) from the nucleus to cytoplasm. We show that nuclear USP7 migrates to the cytoplasm in response to TLR engagement, a process that contributes to termination of TLR response. Cytoplasmic USP7 binds to and deubiquitinates TRAF6 and IKKgamma, thus terminating TLR-mediated NF-kappaB and JNK activation. These findings suggest that USP7 is part of a negative feedback loop regulating TLR signaling and that ICP0 exploits this physiologic process to attenuate innate response to HSV. ICP0 inhibition of the TLR response serves to uncouple the innate and adaptive immune response, thereby playing a key role in HSV pathogenesis and persistence.

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IFITM3 restricts virus-induced inflammatory cytokine production by limiting Nogo-B mediated TLR responses.

Project description:Interferon-induced transmembrane protein 3 (IFITM3) is a restriction factor that limits viral pathogenesis and exerts poorly understood immunoregulatory functions. Here, using human and mouse models, we demonstrate that IFITM3 promotes MyD88-dependent, TLR-mediated IL-6 production following exposure to cytomegalovirus (CMV). IFITM3 also restricts IL-6 production in response to influenza and SARS-CoV-2. In dendritic cells, IFITM3 binds to the reticulon 4 isoform Nogo-B and promotes its proteasomal degradation. We reveal that Nogo-B mediates TLR-dependent pro-inflammatory cytokine production and promotes viral pathogenesis in vivo, and in the case of TLR2 responses, this process involves alteration of TLR2 cellular localization. Nogo-B deletion abrogates inflammatory cytokine responses and associated disease in virus-infected IFITM3-deficient mice. Thus, we uncover Nogo-B as a driver of viral pathogenesis and highlight an immunoregulatory pathway in which IFITM3 fine-tunes the responsiveness of myeloid cells to viral stimulation.

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Neonatal innate TLR-mediated responses are distinct from those of adults.

Project description:The human neonate and infant are unduly susceptible to infection with a wide variety of microbes. This susceptibility is thought to reflect differences from adults in innate and adaptive immunity, but the nature of these differences is incompletely characterized. The innate immune response directs the subsequent adaptive immune response after integrating information from TLRs and other environmental sensors. We set out to provide a comprehensive analysis defining differences in response to TLR ligation between human neonates and adults. In response to most TLR ligands, neonatal innate immune cells, including monocytes and conventional and plasmacytoid dendritic cells produced less IL-12p70 and IFN-alpha (and consequently induced less IFN-gamma), moderately less TNF-alpha, but as much or even more IL-1beta, IL-6, IL-23, and IL-10 than adult cells. At the single-cell level, neonatal innate cells generally were less capable of producing multiple cytokines simultaneously, i.e., were less polyfunctional. Overall, our data suggest a robust if not enhanced capacity of the neonate vs the adult white-blood cell TLR-mediated response to support Th17- and Th2-type immunity, which promotes defense against extracellular pathogens, but a reduced capacity to support Th1-type responses, which promote defense against intracellular pathogens.

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TLR/MyD88-mediated Innate Immunity in Intestinal Graft-versus-Host Disease.

Project description:Graft-versus-host disease (GHVD) is a severe complication after allogeneic hematopoietic stem cell transplantation. The degree of inflammation in the gastrointestinal tract, a major GVHD target organ, correlates with the disease severity. Intestinal inflammation is initiated by epithelial damage caused by pre-conditioning irradiation. In combination with damages caused by donor-derived T cells, such damage disrupts the epithelial barrier and exposes innate immune cells to pathogenic and commensal intestinal bacteria, which release ligands for Toll-like receptors (TLRs). Dysbiosis of intestinal microbiota and signaling through the TLR/myeloid differentiation primary response gene 88 (MyD88) pathways contribute to the development of intestinal GVHD. Understanding the changes in the microbial flora and the roles of TLR signaling in intestinal GVHD will facilitate the development of preventative and therapeutic strategies.

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TLR-4-mediated innate immunity is reduced in cystic fibrosis airway cells.

Project description:Airway epithelial cells contribute to the inflammatory response of the lung, and their innate immune response is primarily mediated via Toll-like receptor (TLR) signaling. Cystic fibrosis (CF) airways are chronically infected with Pseudomonas aeruginosa, suggesting a modified immune response in CF. We investigated the TLR-4 expression and the inflammatory profile (IL-8 and IL-6 secretion) in CF bronchial epithelial cell line CFBE41o- and its CF transmembrane ion condcutance regulator (CFTR)-corrected counterpart grown under air-liquid interface conditions after stimulation with lipopolysaccharide (LPS) from gram-negative bacteria. In CFTR-corrected cells, IL-8 and IL-6 secretions were constitutively activated but significantly increased after LPS stimulation compared with CFBE41o-. Blocking TLR-4 by a specific antibody significantly inhibited IL-8 secretion only in CFTR-corrected cells. Transfection with specific siRNA directed against TLR-4 mRNA significantly reduced the response to LPS in both cell lines. Fluorescence-activated cell sorter analysis revealed significantly higher levels of TLR-4 surface expression in CFTR-corrected cells. In histologic lung sections of patients with CF, the TLR-4 expression in the bronchial epithelium was significantly reduced compared with healthy control subjects. In CF the loss of CFTR function appears to decrease innate immune responses, possibly by altering the expression of TLR-4 on airway epithelial cells. This may contribute to chronic bacterial infection of CF airways.

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The Methanolic Extract of Perilla frutescens Robustly Restricts Ebola Virus Glycoprotein-Mediated Entry.

Project description:Ebola virus (EBOV), one of the most infectious human viruses and a leading cause of viral hemorrhagic fever, imposes a potential public health threat with several recent outbreaks. Despite the difficulties associated with working with this pathogen in biosafety level-4 containment, a protective vaccine and antiviral therapeutic were recently approved. However, the high mortality rate of EBOV infection underscores the necessity to continuously identify novel antiviral strategies to help expand the scope of prophylaxis/therapeutic management against future outbreaks. This includes identifying antiviral agents that target EBOV entry, which could improve the management of EBOV infection. Herein, using EBOV glycoprotein (GP)-pseudotyped particles, we screened a panel of natural medicinal extracts, and identified the methanolic extract of Perilla frutescens (PFME) as a robust inhibitor of EBOV entry. We show that PFME dose-dependently impeded EBOV GP-mediated infection at non-cytotoxic concentrations, and exerted the most significant antiviral activity when both the extract and the pseudoparticles are concurrently present on the host cells. Specifically, we demonstrate that PFME could block viral attachment and neutralize the cell-free viral particles. Our results, therefore, identified PFME as a potent inhibitor of EBOV entry, which merits further evaluation for development as a therapeutic strategy against EBOV infection.

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