1 Department of Microbiology & Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan 70101, Republic of China
2 Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan 70101, Republic of China
3 Department of Pediatrics, College of Medicine, National Cheng Kung University, Tainan, Taiwan 70101, Republic of China
4 Department of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan 70101, Republic of China
5 Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan 70101, Republic of China
Correspondence
Chun-Keung Yu
dckyu{at}mail.ncku.edu.tw
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ABSTRACT |
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INTRODUCTION |
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The induction of type I interferons (IFNs) is the earliest non-specific host response to viral infections (Goodbourn et al., 2000). The type I IFNs IFN-
and IFN-
are produced by leukocytes and fibroblasts, respectively, in response to infection, which leads to the induction of antiviral pathways within hours. Clinically, IFN-
/
has been used for the treatment of hepatitis C virus infection and certain tumours (Kirkwood, 2002
). Sasaki et al. (1986)
showed a therapeutic effect of murine IFN-
/
on coxsackievirus type A16 infection in newborn mice. In this study, we have demonstrated that type I IFNs represent an essential innate defence mechanism for controlling EV71 infection in mice.
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METHODS |
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Mouse experiments.
Specific-pathogen-free, 3-day-old Institute of Cancer Research (ICR) mice (Laboratory Animal Center, National Cheng Kung University, College of Medicine, Tainan, Taiwan) were inoculated intraperitoneally (i.p.) with 100 µl of a pre-determined dose of EV71/Tainan/4643/98 that would result in a 20 or 80 % mortality rate (Yu et al., 2000). Inocula were retitrated by plaque assay immediately after the challenge. Mice were treated with dexamethasone, polyriboinosinic : polyribocytidylic acid [poly(I : C)] (both from Sigma-Aldrich), murine IFN-
/
neutralizing antibody (murine L-cell antiserum; NIAID Reference Reagent Repository) or recombinant mouse IFN-
(R&D Systems). Control mice were given control sera (NIAID Reference Reagent Repository) or PBS. Mice were observed twice daily for clinical illness and death until 21 days of age. Clinical disease was graded as follows: 0, healthy; 1, ruffled fur and hunchbacked appearance; 2, wasting; 3, limb weakness; 4, limb paralysis; and 5, moribund and death. For oral infection experiments, 7-day-old mice were force fed with 200 µl EV71/MP4 using a 24-gauge feeding tube after fasting for 8 h (Wang et al., 2004
). The Institutional Animal Care and Use Committee approved all animal protocols.
Tissue viral titres.
After euthanasia, blood samples were collected following axilla dissection. Blood and serum samples were stored at 70 °C. After perfusion with isotonic saline containing EDTA, tissue samples were aseptically removed, weighed and stored at 70 °C. Tissue samples were homogenized in 1 ml DMEM containing 2 % FBS, disrupted by three freezethaw cycles and centrifuged. Clarified supernatants and blood samples were inoculated onto monolayers of RD cells, which were inspected daily for a minimum of 14 days for cytopathic effect (CPE) (Hsiung, 1994). Viral titres were expressed as log p.f.u. (mg tissue)1 or (ml blood)1. The lower limit of virus detection was 10 p.f.u.
IFN- ELISA.
Concentrations of serum IFN- were measured by a sandwich ELISA technique according to the manufacturer's instruction (Mouse IFN-
ELISA kit; R&D Systems). The detection limit was 7·5 pg ml1.
Flow cytometry for CD11c+ dendritic cells and IFN- expression.
The spleen was removed from mice after euthanasia and mechanically disrupted. Red blood cells were removed into lysing solution containing 20 mM NH4Cl, 10 mM KHCO3 and 0·1 mM EDTA. After washing, a single-cell suspension (106 cells in 1 ml Hanks' balanced salt solution containing 2 % fetal calf serum and 0·1 % sodium azide) was incubated with anti-CD16/CD32 mAb (Fc blocker, 2.4G2; BD PharMingen), followed by fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (H129.19; BD PharMingen) and phycoerythrin (PE)-conjugated anti-CD8a (53-6.7; BD PharMingen), PECy5-conjugated anti-major histocompatibility complex (MHC) class II (M5/114.15.2; eBioscience) or PECy7-conjugated anti-B220 (RA3-6B2; eBioscience) mAbs for 30 min on ice. After washing, stained cells were quantified using flow cytometry (BD Immunocytometry Systems). Isotype-matched mAb-stained cells were used as a background control in all experiments. For intracellular cytokine staining, single-cell suspensions of splenocytes were stimulated with 10 µg phorbol myristate acetate ml1 plus 1 µM ionomycin (Sigma-Aldrich) in the presence of monensin (eBioscience) for 5 h in six-well plates. Cells (106 ml1) were first stained with PE-conjugated anti-CD11c (N418; eBioscience), followed by anti-mouse IFN- mAb (R&D Systems) and secondary antibody after fixation and permeabilization. Cells were then analysed using flow cytometry as described above.
In vitro CPE protection assay.
RD, Caco-2 or SK-N-SH cells (5x105 cells per well) were plated on 24-well plates, grown overnight to obtain >80 % confluence and then primed with human leukocyte IFN-/
(R&D Systems) for 6 h or left untreated. After washing, the monolayers were infected with EV71/Tainan/4643/98 at an m.o.i. of 0·001. When 90 % of the cells in the non-treated wells had CPE, the wells were stained with 0·5 % crystal violet. In other experiments, RD or SK-N-SH cells (2x104 cells per well) were plated on 96-well plates and cultured for 18 h before treatment with increasing doses of IFN-
/
(final concentrations ranging from 0 to 2500 U ml1) for 6 h. After washing, the monolayers were infected with EV71/Tainan/4643/98 (dilutions of 101 to 109). CPE was examined after incubation for 72 h and TCID50 values were determined by the method of Hsiung (1994)
using the Reed & Muench formula.
Statistical analysis.
The frequencies of survival and mortality in treated mice versus control mice were assessed using Fisher's exact two-tailed test. Differences in the mean survival time, tissue viral titres, serum IFN- concentrations and TCID50 were determined using a two-tailed MannWhitney U test or KruskalWallis test. Values were considered significant for values of P<0·05.
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RESULTS |
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DISCUSSION |
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The host factors that influence whether infection with EV71 culminates in a terminal disease or a mild infection state are not known (Chang et al., 1999). Alteration of cellular immunity of the hosts has been suggested to be related to disease severity (Yang et al., 2001
). Immunodeficient mice including nude, B-cell deficient, SCID, CD28 knockout and tumour necrosis factor-
receptor knockout mice have been used to delineate the role of the immune system in EV71 infection. However, none of these mouse strains was more susceptible to infection than the wild type (Wu et al., 2002
). On the other hand, we observed that nitric oxide might represent an important innate immunity for EV71 infection in our murine infection model (P. W. Kuo & J. R. Wang, unpublished observation). Since the type I IFN system is the most important innate defence against viral infection, we next attempted to define the role of type I IFNs in EV71 infection. Poly(I : C) is a potent IFN inducer and poly(I : C) administration resulted in a rapid accumulation of high concentrations of IFN at the local site of virus replication, which acted in an autocrine manner (Poast et al., 2002
; Pyo et al., 1993
). Therefore, we first tested the prophylactic effect of poly(I : C). We demonstrated that poly(I : C) augmented IFN-
production and activated splenic IFN-
-producing dendritic cells in mice. Furthermore, poly(I : C) not only improved the survival rate but also decreased the viral titre in the brain tissue of EV71-infected mice. Similarly, Padalko et al. (2004)
showed that poly(I : C) could markedly protect mice from coxsackie B3 virus-induced mycocarditis. Recent studies have shown that poly(I : C) can activate natural killer (NK) cells (Schmidt et al., 2004
) and
T cells via type I IFNs derived from toll-like receptor-3-expressing CD11c+ dendritic cells (Kunzmann et al., 2004
). Thus, the observed protective effect of poly(I : C) might be attributed to the activation of CD11c+ dendritic cells, NK cells and
T cells, and the production of IFN-
. Poly(I : C) has been given intravenously to patients with viral infection, but the low titres and short duration of IFN induced [8500 U ml1 in response to doses of poly(I : C) of 0·11·0 mg kg1] might limit its clinical usefulness as an antiviral drug, as stated by Guggenheim & Baron (1977)
. In contrast to poly(I : C), EV71 inoculation was unable to elicit the production of type I IFNs. More studies are needed to clarify whether EV71 exerts any inhibitory effect on the production of type I IFNs.
Although EV71 inoculation did not elicit an increase in serum type I IFNs, two series of in vivo experiments confirmed the protective role of type I IFNs on EV71 infection. First, pre-treatment with a neutralizing antibody to IFN-/
dramatically increased the susceptibility of mice to EV71. Secondly, the therapeutic effect of recombinant murine IFN-
A was clearly demonstrated in mice infected by either the i.p. or the oral route. An early treatment of recombinant IFN-
was essential for the outcome of the infection, as no antiviral activity was observed once neuroinvasion was evident at 3 days p.i. EV71-infected mice developed clinical symptoms around 4 or 5 days p.i. IFN treatment at this time point may offer no benefit but may worsen the infection, which may explain why the survival rate of IFN-treated mice (treated at 3, 4 and 5 days p.i.) was lower in comparison with infected non-treated control mice. Furthermore, in vitro experiments demonstrated that human IFN-
/
could protect against EV71 infection of the three major target cell types of EV71, namely RD, Caco-2 and SK-N-SH cells.
Collectively, our data indicate that type I IFNs play an important role in the host defence of EV71 and that an earlier detection of viral involvement of the central nervous system followed by immediate type I IFN therapy should be considered an appropriate treatment regimen for EV71.
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ACKNOWLEDGEMENTS |
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Received 21 May 2005;
accepted 22 August 2005.
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