Stephanie J. DeWitte-Orr

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.

Long Double-Stranded RNA Induces an Antiviral Response Independent of IFN Regulatory Factor 3, IFN-β Promoter Stimulator 1, and IFN

Virus infection elicits a robust innate antiviral response dominated by the production of type 1 IFN. In nonprofessional innate immune cells such as fibroblasts, type 1 IFN is rapidly produced following the recognition of viral dsRNA and the subsequent activation of the constitutively expressed transcription factor IFN regulatory factor 3 (IRF3). Although origin, localization, and length are factors in mediating dsRNA recognition and binding by cellular dsRNA-binding proteins, the biological significance of differential dsRNA binding is unclear, since the subsequent signaling pathways converge on IRF3. In this study, we show a dsRNA length-dependent activation of IRFs, IFNs, and IFN-stimulated genes in mouse fibroblasts. The length dependence was exacerbated in fibroblasts deficient in the mitochondria-associated adaptor IFN-β promoter stimulator 1 and IRF3, suggesting that antiviral gene induction mediated by short and long dsRNA molecules is predominantly IFN-β promoter stimulator 1 and IRF3 dependent and independent, respectively. Furthermore, we provide evidence of an innate antiviral response in fibroblasts in the absence of both IRF3 and type 1 IFN induction. Even with these key modulators missing, a 60–90% inhibition of virus replication was observed following 24-h treatment with short or long dsRNA molecules, respectively. These data provide evidence of a novel antiviral pathway that is dependent on dsRNA length, but independent of the type 1 IFN system.

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-kappaB (NF-kappaB) 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-beta stimulation. This was associated with decreased nuclear translocation of phosphorylated Stat1 in response to IFN-beta 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.

Sensors of Infection: Viral Nucleic Acid PRRs in Fish

Viruses produce nucleic acids during their replication, either during genomic replication or transcription. These nucleic acids are present in the cytoplasm or endosome of an infected cell, or in the extracellular space to be sensed by neighboring cells during lytic infections. Cells have mechanisms of sensing virus-generated nucleic acids; these nucleic acids act as flags to the cell, indicating an infection requiring defense mechanisms. The viral nucleic acids are called pathogen-associated molecular patterns (PAMPs) and the sensors that bind them are called pattern recognition receptors (PRRs). This review article focuses on the most recent findings regarding nucleic acids PRRs in fish, including: Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), cytoplasmic DNA sensors (CDSs) and class A scavenger receptors (SR-As). It also discusses what is currently known of the downstream signaling molecules for each PRR family and the resulting antiviral response, either type I interferons (IFNs) or pro-inflammatory cytokine production. The review highlights what is known but also defines what still requires elucidation in this economically important animal. Understanding innate immune systems to virus infections will aid in the development of better antiviral therapies and vaccines for the future.

A critical role for IL-15 in TLR-mediated innate antiviral immunity against genital HSV-2 infection

Innate antiviral immunity, particularly at mucosal surfaces, has a critical role in early control of viral infections. Both type I interferons (IFNs) and interleukin‐15 (IL‐15) are essential components of innate antiviral immunity. It has been shown that toll‐like receptor (TLR) ligand‐induced innate antiviral immunity requires IFN‐α/β and ‐λ receptor signaling. However, it is not known if IL‐15 has a role in TLR ligand‐mediated antiviral responses. Here, we report that ligands for TLR‐3 and TLR‐9 cannot confer protection against genital herpes simplex virus‐2 (HSV‐2) in the absence of IL‐15 in vivo. Interestingly, wild‐type mice depleted of natural killer (NK) cells and treated with TLR ligands are protected upon HSV‐2 challenge, suggesting that the critical role of IL‐15 is independent of NK cell‐mediated activity. To examine the cytokine response in the absence of IL‐15, we investigated TLR ligand‐induced IFN‐β and ‐λ production in the vaginal washes, but found no impairment in IL‐15−/− mice. Finally, we report no impairment in the expression of the IFN‐stimulated genes in IL‐15−/− mice. Collectively, the data suggest that TLR ligands induce an IFN‐mediated response in the vaginal tract of both wild‐type and IL‐15−/− mice, but its induction is insufficient for providing protection against HSV‐2 in the absence of IL‐15.

A comparison of rainbow trout cell lines for their expression of the major histocompatibility complex genes and the induction of beta-2-microglobulin by dsRNA

Western blotting with polyclonal antisera to polypeptides of the rainbow trout major histocompatibility (MH) genes and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to compare expression of MH genes in rainbow trout cell lines. One line was the spleen monocyte/macrophage-like RTS11, which grew loosely on plastic surfaces. Adherent cell lines were fibroblast-like RTG-2 from gonads and four epithelial-like cell lines from gill, intestine, liver and hepatoma: RTgill-W1, RTgutGC, RTL-W1, and RTH-149 respectively. All cell lines expressed a 45 kDa MHC class I alpha chain. All cell lines expressed beta-2-microglobulin (beta2m), which was at 11 kDa, but detection was abrogated following trypsinization prior to cell collection. All cell lines expressed transcripts for MH class II alpha and MH class II beta genes; however, MH class II polypeptides were expressed only in RTS11, the only cell line from a lineage of antigen-presenting cells. We report here that double stranded RNA up regulates beta2m and that these cell lines and antisera can be employed for studying MH regulation.

Development of a continuous cell line, PBLE, from an American eel peripheral blood leukocyte preparation.

SummaryA continuous cell line, PBLE, was developed from the adherent cells in a culture of peripheral blood leukocytes from the American eel, Anguilla rostrata. The cells were grown in Leibovitzs L-15 basal medium supplemented with 20% fetal bovine serum (FBS). Under normal culture conditions at 18° C, the morphology of PBLE was fibroblast-like. The cultures have been subcultured over 80 times and have been cryopreserved successfully. These cells have a diploid karyotype of 38 chromosomes, survived temperatures from 5 to 36° C, and proliferated at temperatures from 5° C to at least 30° C. PBLE underwent apoptosis in response to gliotoxin, but did not show a respiratory burst. Results suggest that PBLE may have arisen from a circulating mesenchymal stem cell. PBLE was susceptible to Chum salmon reovirus (CSV) and supported CSV replication. Therefore this cell line should be useful in studying eel specific virus-host interactions.

Fish interferon-stimulated genes: The antiviral effectors.

Type I interferons (IFN) are the cornerstone cytokine of innate antiviral immunity. In response to a viral infection, IFN signaling results in the expression of a diverse group of genes known as interferon-stimulated genes (ISGs). These ISGs are responsible for interfering with viral replication and infectivity, helping to limit viral infection within a cell. In mammals, many antiviral effector ISGs have been identified and the antiviral mechanisms are at least partially elucidated. In fish fewer ISGs have been identified and while there is evidence they limit viral infection, almost nothing is known of their respective antiviral mechanisms. This review discusses seven ISGs common to mammals and fish and three ISGs that are unique to fish. The lack of understanding regarding fish ISGs antiviral effector functions is highlighted and draws attention to the need for research in this aspect of aquatic innate immunity.

Class-A scavenger receptor function and expression in the rainbow trout (Oncorhynchus mykiss) epithelial cell lines RTgutGC and RTgill-W1.

Class A scavenger receptors (SR-As) are cell surface receptors that bind a range of ligands, including modified low-density lipoproteins (mLDLs) and nucleic acids. Due to their ability to bind extracellular dsRNA, SR-As play an important role in the viral dsRNA initiated immune pathway. Most research on SR-As has focused on mammalian models, and there has been limited research on SR-As in fish. Thus, the presence of functional class A scavenger receptors (SR-As) were investigated in the rainbow trout cell lines, RTgutGC and RTgill-W1. SR-A ligand binding was assessed using fluorescently labeled acetylated-low density lipoprotein (acLDL) and synthetic dsRNA, polyinosinic:polycytidylic acid (poly IC), in combination with a series of known SR-A competitive ligands: fucoidan, dextran sulfate (DxSO4) and polyinosinic acid (poly I). Both cell lines were able to bind acLDL, which was blocked by SR-A competitive ligands. In RTgutGC, acLDL and poly IC competed for binding to the same surface receptor; however, in RTgill-W1 they did not. Poly IC-fluorescein binding was blocked by SR-A competitive ligands in RTgutGC but not RTgill-W1, suggesting an SR-A dependent dsRNA uptake mechanism in RTgutGC and an SR-A-independent update mechanism in RTgill-W1. Both cell lines responded to extracellular dsRNA treatment with the up-regulation of interferons (IFNs) and interferon stimulated genes (ISGs) as measured by quantitative (q)RT-PCR; however, RTgutGC expressed significantly higher transcript levels for both IFNs and ISGs compared with RTgill-W1 following extracellular poly IC treatment. Expression of SR-As, specifically a SCARA4-like sequence, was identified at the transcript level in both cell lines. These results suggest that both RTgill-W1 and RTgutGC express functional SR-As that are able to bind the classic SR-A ligand, acLDL. Although they both express SCARA4, the full SR-A expression profile; however, is likely different between the cell lines, as dsRNA uptake appears to be SR-A dependent in RTgutGC but SR-A-independent in RTgill-W1. Also, dsRNA uptake via SR-As appears to mediate a more robust antiviral response compared with a SR-A independent method of uptake. This study is the first to identify functional SR-As in rainbow trout epithelial cells, and contributes not only to a better understanding of modified LDL transport but also innate immunity in these economically important animals.

Length-dependent innate antiviral effects of double-stranded RNA in the rainbow trout (Oncorhynchus mykiss) cell line, RTG-2.

Effectively all viruses produce long dsRNA during their replicative cycle. In mammals long dsRNA molecules induce a robust response through the production of type 1 interferon, interferon-stimulated genes (ISGs) and an antiviral state. This response is less well understood in fish. We investigated the ability of a rainbow trout cell line, RTG-2, to respond to two different lengths of in vitro transcribed dsRNA (200 bp and 1264 bp) based on the viral hemorrhagic septicemia virus genomic sequence, and high and low molecular weight poly I:C (synthetic dsRNA). To explore the innate immune response we used qRT-PCR to measure immune gene transcript levels, an ISG-promoter reporter assay, and an antiviral protection assay. We saw a significantly greater immune response in all assays in response to the longer dsRNA molecule compared to their shorter counterpart. We saw significantly more interferon and ISG transcripts, stronger induction of a protective antiviral state, and more robust activation of the ISG-promoter. This response was not found to be due to a better uptake of the longer dsRNA molecules as a cellular uptake assay showed no differences between lengths. These data suggest that dsRNA-mediated innate immune responses are length-dependent and longer molecules induce a more robust response. There were also some differences in the cells response to in vitro transcribed dsRNA compared to poly I:C. This provides important information for potential dsRNA-based antiviral therapies and vaccine adjuvants.