The viral dsRNA analogue poly (I:C) induces necrotizing enterocolitis in neonatal mice.

BACKGROUND: Necrotizing enterocolitis (NEC) is a life-threatening gastrointestinal disease in premature infants with high mortality and morbidity with uncertain pathogenesis. Recent research focused on the role of intraluminal bacteria and lipopolysaccharide (LPS). However, an additional role of viral agents in the pathogenesis of NEC has recently been postulated. We assessed the role of polyinosinic:polycytidylic acid (pIC) mimicking viral dsRNA in contributing to the development of NEC in neonatal mice. METHODS: Four-d-old C57BL/6J pups were stressed by asphyxia and hypothermia twice daily. Animals were either fed by formula only (FO), formula containing LPS or pIC. After 72 h, mice were euthanized, intestines harvested, and the severity of NEC was assessed. RESULTS: Breastfed mice showed no evidence of NEC. Very mild NEC-like lesions were observed in mice fed by FO. Supplementation of LPS or pIC to the formula led to increased intestinal tissue damage and inflammation compared with FO in a similar manner. CONCLUSION: Our study demonstrates the ability of viral factors to induce NEC in neonatal mice even in the absence of LPS. Furthermore, we present a new mouse model of pIC-induced NEC which may be used to obtain further mechanistic insights in the pathogenesis of this disease.

TLR3, TRIF, and caspase 8 determine double-stranded RNA-induced epithelial cell death and survival in vivo.

TLR3 signaling is activated by dsRNA, a virus-associated molecular pattern. Injection of dsRNA into mice induced a rapid, dramatic, and reversible remodeling of the small intestinal mucosa with significant villus shortening. Villus shortening was preceded by increased caspase 3 and 8 activation and apoptosis of intestinal epithelial cells (IECs) located in the mid to upper villus with ensuing luminal fluid accumulation and diarrhea because of an increased secretory state. Mice lacking TLR3 or the adaptor molelcule TRIF mice were completely protected from dsRNA-induced IEC apoptosis, villus shortening, and diarrhea. dsRNA-induced apoptosis was independent of TNF signaling. Notably, NF-κB signaling through IκB kinase ß protected crypt IECs but did not protect villus IECs from dsRNA-induced or TNF-induced apoptosis. dsRNA did not induce early caspase 3 activation with subsequent villus shortening in mice lacking caspase 8 in IECs but instead caused villus destruction with a loss of small intestinal surface epithelium and death. Consistent with direct activation of the TLR3-TRIF-caspase 8 signaling pathway by dsRNA in IECs, dsRNA-induced signaling of apoptosis was independent of non-TLR3 dsRNA signaling pathways, IL-15, TNF, IL-1, IL-6, IFN regulatory factor 3, type I IFN receptor, adaptive immunity, as well as dendritic cells, NK cells, and other hematopoietic cells. We conclude that dsRNA activation of the TLR3-TRIF-caspase 8 signaling pathway in IECs has a significant impact on the structure and function of the small intestinal mucosa and suggest signaling through this pathway has a host protective role during infection with viral pathogens.

Double-stranded RNA exacerbates pulmonary allergic reaction through TLR3: implication of airway epithelium and dendritic cells.

Respiratory viral infections have been implicated in exacerbations of allergic asthma, characterized by a Th2-biased immune response. Respiratory viruses target airway epithelial cells and dendritic cells (DCs). Their activation is, at least in part, mediated by the TLR3-dependent recognition of virus-derived dsRNA. To elucidate the role of epithelial cells and DCs and the implication of TLR3/Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF) pathway, we developed a mouse model of lung allergic exacerbation. The effect of intranasal administration of dsRNA in OVA-sensitized wild-type mice and TRIF(-/-) mice was evaluated on airway hyperresponsiveness and pulmonary inflammation. Our data demonstrated that treatment with dsRNA significantly increased the airway hyperresponsiveness, the lung inflammation, and the OVA-specific Th2 response. This was associated with an infiltrate of eosinophils, myeloid DCs, and T lymphocytes. TRIF activation was required for the development of dsRNA-induced exacerbation of the allergic reaction. Intratracheal transfer of IL-4/dsRNA/OVA-pretreated DCs also triggered exacerbation of the allergic reaction, whereas cells primed with dsRNA/OVA had a more limited effect. dsRNA-induced production of CCL20 by airway epithelium was associated with DC recruitment. In vivo and in vitro treatment with dsRNA amplified airway epithelial production of the pro-Th2 chemokines CCL11 and CCL17, their secretion being enhanced by Th2 cytokines. In conclusion, dsRNA derived from respiratory viruses trigger exacerbation of the pulmonary allergic reaction through TLR3/TRIF-dependent pathway. Moreover, Th2 cytokines participate in this process by modulating the response of airway epithelium and DCs to dsRNA.

Chronic cigarette smoke exposure primes NK cell activation in a mouse model of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a debilitating, progressive lung disease punctuated by exacerbations of symptoms. COPD exacerbations are most often associated with viral infections, and exposure to cigarette smoke (CS) followed by viral infection has been shown experimentally to enhance lung inflammation, tissue destruction, and airway fibrosis. Despite this, however, the cellular mechanisms responsible for this effect are unknown. In this study, we examined NK cell function in a mouse model of COPD given the vital role of NK cells following viral infection. Ex vivo stimulation of lung leukocytes with poly(I:C), ssRNA40, or ODN1826 enhanced production of NK cell-derived IFN-gamma in CS-exposed mice. NK cells from CS-exposed mice exhibited a novel form of priming; highly purified NK cells from CS-exposed mice, relative to NK cells from filtered air-exposed mice, produced more IFN-gamma following stimulation with IL-12, IL-18, or both. Further, NK cell priming was lost following smoking cessation. NKG2D stimulation through overexpression of Raet1 on the lung epithelium primed NK cell responsiveness to poly(I:C), ssRNA40, or ODN1826 stimulation, but not cytokine stimulation. In addition, NK cells from CS-exposed mice expressed more cell surface CD107a upon stimulation, demonstrating that the NK cell degranulation response was also primed. Together, these results reveal a novel mechanism of activation of the innate immune system and highlight NK cells as important cellular targets in controlling COPD exacerbations.

Arthritogenic properties of double-stranded (viral) RNA.

Viral infections often lead to arthralgias and overt arthritic states. The inflammatogenic compound of the viruses giving rise to such an outcome has to date not been identified. Because expression of dsRNA is a common feature of all viruses, we decided to analyze whether this property leads to the induction of arthritis. Histological signs of arthritis were evident already on day 3 following intra-articular administration of dsRNA. Arthritis was characterized by infiltration of macrophages into synovial tissue. It was not dependent on acquired immune responses because SCID mice also raised joint inflammation. NF-kappa B was activated upon in vitro exposure to dsRNA, indicating its role in the induction/progression of arthritis. Importantly, we found that dsRNA arthritis was triggered through IL-1R signaling because mice being deficient for this molecule were unable to develop joint inflammation. Although dsRNA is typically recognized by Toll-like receptor 3, Toll-like receptor 3 knockout mice developed arthritis, indicating that some other receptors are instrumental in the inducing of inflammation. Our results from in vitro experiments indicate that proinflammatory cytokines and chemokines stimulating monocyte influx were readily triggered in response to stimulation with dsRNA. These findings demonstrate that viral dsRNA is clearly arthritogenic. Importantly, macrophages and their products play an important role in the development of arthritis triggered by dsRNA.

Recovery of infectious foot-and-mouth disease virus from suckling mice after direct inoculation with in vitro-transcribed RNA.

We assayed the infectivity of naked foot-and-mouth disease virus (FMDV) RNA by direct inoculation of suckling mice. Our results demonstrate that transcripts generated from full-length cDNA clones were infectious, as was virion-extracted RNA. Interestingly, infectious virus could be recovered from a mutant transcript encoding amino acid substitution L-147-->P in capsid protein VP1, known to be noninfectious for BHK-21 cells. The model described here provides a useful tool for virulence studies in vivo, bypassing possible selection of variants during viral replication in cell culture.

A candidate Ross River virus vaccine: preclinical evaluation.

A killed Ross River virus vaccine is being developed in an effort to prevent the, ca 5000 cases of epidemic polyarthritis which occur in Australia each year. The symptoms of epidemic polyarthritis commonly last 30-40 weeks and 25% of patients have symptoms for a year or more. There is no cure. Although there was some strain to strain variation, particularly after a single injection, outbred and inbred strains of mice all produced significant levels of anti-Ross River virus antibody after intramuscular (i.m.) injection with 24 h BEI inactivated, sucrose gradient purified, Ross River virus vaccine. Mice immunized i.m. with two 20 micrograms doses of vaccine or live virus produced similar levels of neutralizing antibody but the reaction of IgG 2a and IgG 2b antibody from these two groups of mice to Ross River virus proteins in western blots differed. Antibody from BALB/c mice immunized with this vaccine neutralized all strains of Ross River virus tested, in vitro, albeit to different degrees.

Infectivity of linear monomeric transcripts of citrus exocortis viroid: terminal sequence requirements for processing.

Infectivity of linear monomeric viroid RNA transcripts is known to be dependent on the site of cloning of the viroid and the viroid and vector sequences at the termini of the RNA transcripts. We report here the further characterization of cloning site and sequence requirements using 11 different monomeric cDNA constructs of citrus exocortis viroid; these differed from one another by the site of cloning and by nonviroid residues adjacent to the 5' and 3' termini of the viroid sequence in the RNA transcript. The infectivity of these transcripts was assayed by inoculation onto tomato seedlings; all infectious transcripts were wild type, indicating accurate processing. The basic requirement for infectivity appeared to be the ability of the RNA transcripts to form a short double-stranded region of viroid and vector sequences at the junction of the two termini. The site of cloning of the viroid RNA used for cloning was not important as cDNA clones prepared at seven sites provided infectious transcripts.

[Comparative study of interferon inducers obtained from natural sources]. / Sravnitel'noe issledovanie induktorov interferona, poluchennykh iz prirodnykh istochnikov.

Interferon-inducing and antiviral effects of natural dsRNA preparations of phage phi 6 and yeast cells were studied in the culture of murine cells L-929 and on random bred albino mice. Both the preparations showed interferon inducing activity in the cell culture. However, for realization of their effect modification of the surface cell membrane by polycation exchange resin (DEAE-dextran) was required. The interferon-inducing activity of both of the natural dsRNA in the mice was high. The maximum interferon titers (1280-5120 units/ml) in blood serum were observed 4-6 hours after the inductor intraperitoneal administration. The interferon-inducing activity of the phage dsRNA was high in the cell culture and yeast dsRNA--in mice, respectively. Both the inductors had antiviral activity and protected 15 to 38.9 per cent of the experimental animals from the effect of 100 LD50 of the murine encephalomyocarditis virus and 10 LD50 of the influenza virus A/Aichi 2/68 (H3N2).

[Properties of double-stranded RNA as an interferon inducer]. / Svoistva dvuspiral'noi RNK kak induktora interferona.

The stability and activity of double-stranded RNA (dsRNA) preparations as interferon inductors were studied. They were found to be stable upon long storage and ultrasonic treatment for aerosol preparation. Data on dsRNA toxicity for mice and rabbits are presented. The kinetics of interferon production and its distribution in tissues in relation to the routes of inoculation were studied. Investigation of dsRNA antiviral activity in various experimental viral infections demonstrated a protective effect of the preparation after a single prophylactic administration of the inductor.

Use of bacteriophage nucleic acid inducers of interferon in man.

The interest in therapeutic exploration of phage ds-RNAs is motivated by their antiviral and immunoregulatory activities. Preliminary studies in man indicate that topical application of native ds-RNAs represents no health-hazard to patients and may be beneficial in the therapy of viral skin and eye diseases.

Antiviral effects of double-stranded RNA from rice dwarf virus on infection of mice with western equine encephalitis virus.

A single administration (1 to 10 mg/kg) of rice dwarf virus RNA (RDV-RNA) prior to virus challenge reduced the mortality of mice infected with western equine encephalitis (WEE) virus. The protective effect of RDV-RNA was significantly higher than that of polyinosinic-polycytidylic acid. However, when these RNAs were given after virus infection, the protective effect was negligible. The titer of circulating interferon in mice reached a peak about 5 hr after injection of these RNAs and remained at this level for about 24 hr. Viremia in mice infected with a lethal dose of WEE virus was markedly suppressed by the treatment of mice with these RNAs. A pathological examination of mice treated with a lethal dose of RDV-RNA revealed marked changes including degeneration and karyorrhexis in the lymphoid tissues of the spleen.

Increased resistance of an interferon-resistant cell subline to the toxic effect of double-stranded RNA.

The toxicity of double-stranded (ds) f2-phage RNA for interferon-treated L cells and an interferon-resistant subline of L cells (CVS cells) was compared. The interferon-resistant subline proved to be about 20 times more resistant to ds RNA than the parent L cells. The degree of toxicity of ds RNA was dependent on the concentration of interferon to which the cells were exposed.

Increased toxicity of double-stranded ribonucleic acid in virus-infected animals.

Virus-infected mice were significantly more susceptible to the toxic effects of double-stranded ribonucleic acid (RNA) than uninfected mice. A dramatic increase in mortality was observed after injection of either synthetic (polyriboinosinic.polyribocytidylic acid) or natural (mycophage) double-stranded RNA in mice infected with Newcastle disease virus (NDV) or vesicular stomatitis virus (VSV). With the exception of endotoxin, interferon inducers other than double-stranded RNA, such as tilorone-hydrochloride and chlorite-oxidized oxyamylose, did not show this increased toxicity in virus-infected animals. The increased susceptibility of virus-infected animals to the toxic effects of double-stranded RNA appears to be related to the levels of interferon induced by the virus infection, either systemically, in the blood stream (after inoculation of NDV), or locally, in the brain (after infection with VSV).