Cigarette Smoke Suppresses Type I Interferon-Mediated Antiviral Immunity in Lung Fibroblast and Epithelial Cells

The objective of this study was to investigate the impact of cigarette smoke on innate antiviral defense mechanisms; specifically, we examined the effects of cigarette smoke on the induction of type I interferon (IFN). We observed a dose-dependent decrease in the ability of human lung fibroblast and epithelial cells to elicit an antiviral response against a viral double-strand RNA (dsRNA) mimic, polyI:C, in the presence of cigarette smoke-conditioned medium (SCM). Mechanistically, SCM decreases the expression of IFN-stimulated gene 15 (ISG15) and IFN regulatory factor-7 (IRF-7) transcripts and suppresses the nuclear translocation of key transcription factors, nuclear factor-κB (NF-κB) and IRF-3, after polyI:C stimulation. Furthermore, we provide evidence that the intercellular defense strategy against viral infection is also impaired. We observed a decrease in the ability of fibroblasts to elicit an antiviral state in response to IFN-β stimulation. This was associated with decreased nuclear translocation of phosphorylated Stat1 in response to IFN-β treatment. The effects elicited by SCM are reversible and are almost entirely abrogated in the presence of an antioxidant, such as glutathione. Our findings suggest that cigarette smoke affects the immediate-early, inductive, and amplification phases of the type I IFN response

Extracellular dsRNA induces a type I interferon response mediated via class A scavenger receptors in a novel Chinook salmon derived spleen cell line

The final publication is available at Elsevier via https://dx.doi.org/10.1016/j.dci.2018.08.010 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/Despite increased global interest in Chinook salmon aquaculture, little is known of their viral immune defenses. This study describes the establishment and characterization of a continuous cell line derived from Chinook salmon spleen, CHSS, and its use in innate immune studies. Optimal growth was seen at 14–18 °C when grown in Leibovitz's L-15 media with 20% fetal bovine serum. DNA analyses confirmed that CHSS was Chinook salmon and genetically different from the only other available Chinook salmon cell line, CHSE-214. Unlike CHSE-214, CHSS could bind extracellular dsRNA, resulting in the rapid and robust expression of antiviral genes. Receptor/ligand blocking assays confirmed that class A scavenger receptors (SR-A) facilitated dsRNA binding and subsequent gene expression. Although both cell lines expressed three SR-A genes: SCARA3, SCARA4, and SCARA5, only CHSS appeared to have functional cell-surface SR-As for dsRNA. Collectively, CHSS is an excellent cell model to study dsRNA-mediated innate immunity in Chinook salmon.Natural Sciences and Engineering Research Council of CanadaCanada Research Counci

An Accessory to the ‘Trinity’: SR-As Are Essential Pathogen Sensors of Extracellular dsRNA, Mediating Entry and Leading to Subsequent Type I IFN Responses

Extracellular RNA is becoming increasingly recognized as a signaling molecule. Virally derived double stranded (ds)RNA released into the extracellular space during virus induced cell lysis acts as a powerful inducer of classical type I interferon (IFN) responses; however, the receptor that mediates this response has not been identified. Class A scavenger receptors (SR-As) are likely candidates due to their cell surface expression and ability to bind nucleic acids. In this study, we investigated a possible role for SR-As in mediating type I IFN responses induced by extracellular dsRNA in fibroblasts, a predominant producer of IFNβ. Fibroblasts were found to express functional SR-As, even SR-A species thought to be macrophage specific. SR-A specific competitive ligands significantly blocked extracellular dsRNA binding, entry and subsequent interferon stimulated gene (ISG) induction. Candidate SR-As were systematically investigated using RNAi and the most dramatic inhibition in responses was observed when all candidate SR-As were knocked down in unison. Partial inhibition of dsRNA induced antiviral responses was observed in vivo in SR-AI/II-/- mice compared with WT controls. The role of SR-As in mediating extracellular dsRNA entry and subsequent induced antiviral responses was observed in both murine and human fibroblasts. SR-As appear to function as ‘carriers’, facilitating dsRNA entry and delivery to the established dsRNA sensing receptors, specifically TLR3, RIGI and MDA-5. Identifying SR-As as gatekeepers of the cell, mediating innate antiviral responses, represents a novel function for this receptor family and provides insight into how cells recognize danger signals associated with lytic virus infections. Furthermore, the implications of a cell surface receptor capable of recognizing extracellular RNA may exceed beyond viral immunity to mediating other important innate immune functions

Understanding Viral dsRNA-Mediated Innate Immune Responses at the Cellular Level Using a Rainbow Trout Model

Viruses across genome types produce long dsRNA molecules during replication [viral (v-) dsRNA]. dsRNA is a potent signaling molecule and inducer of type I interferon (IFN), leading to the production of interferon-stimulated genes (ISGs), and a protective antiviral state within the cell. Research on dsRNA-induced immune responses has relied heavily on a commercially available, and biologically irrelevant dsRNA, polyinosinic:polycytidylic acid (poly I:C). Alternatively, dsRNA can be produced by in vitro transcription (ivt-) dsRNA, with a defined sequence and length. We hypothesized that ivt-dsRNA, containing legitimate viral sequence and length, would be a more appropriate proxy for v-dsRNA, compared with poly I:C. This is the first study to investigate the effects of v-dsRNA on the innate antiviral response and to compare v-dsRNA to ivt-dsRNA-induced responses in fish cells, specifically rainbow trout. Previously, class A scavenger receptors (SR-As) were found to be surface receptors for poly I:C in rainbow trout cells. In this study, ivt-dsRNA binding was blocked by poly I:C and v-dsRNA, as well as SR-A competitive ligands, suggesting all three dsRNA molecules are recognized by SR-As. Downstream innate antiviral effects were determined by measuring IFN and ISG transcript levels using qRT-PCR and antiviral assays. Similar to what has been shown previously with ivt-dsRNA, v-dsRNA was able to induce IFN and ISG transcript production between 3 and 24 h, and its effects were length dependent (i.e., longer v-dsRNA produced a stronger response). Interestingly, when v-dsRNA and ivt-dsRNA were length and sequence matched both molecules induced statistically similar IFN and ISG transcript levels, which resulted in similar antiviral states against two aquatic viruses. To pursue sequence effects further, three ivt-dsRNA molecules of the same length but different sequences (including host and viral sequences) were tested for their ability to induce IFN/ISG transcripts and an antiviral state. All three induced responses similarly. This study is the first of its kind to look at the effects v-dsRNA in fish cells as well as to compare ivt-dsRNA to v-dsRNA, and suggests that ivt-dsRNA may be a good surrogate for v-dsRNA in the study of dsRNA-induced responses and potential future antiviral therapies

dsRNA and the innate antiviral immune response

Innate immunity is the first line of defense against viral infections. It is based on a mechanism of sensing pathogen-associated molecular patterns through host germline-encoded pattern recognition receptors. dsRNA is arguably the most important viral pathogen-associated molecular pattern due to its expression by almost all viruses at some point during their replicative cycle. Viral dsRNA has been studied for over 55 years, first as a toxin, then as a type I interferon inducer, a viral mimetic and an immunomodulator for therapeutic purposes. This article will focus on dsRNA, its structure, generation (both endogenous and viral), host sensing mechanisms and induction of type I interferons. The possible therapeutic applications of these findings will also be discussed. The goal of this article is to give an overview of these mechanisms, highlighting novel findings, while providing a historical perspective. </jats:p