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Short Communication |
Plum Island Animal Disease Center, USDA-ARS, PO Box 848, Greenport, NY 11944, USA
Correspondence
C. L. Afonso
claudio.afonso{at}ars.usda.gov
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ABSTRACT |
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. Lower levels of nitric oxide and increased arginase activity were found in CSFV-infected macrophages. These changes in gene expression in macrophages suggest viral modulation of host expression to suppress nitric oxide production.
Present address: USDA/ARS/SEPRL, 934 College Station Road, Athens, GA 30605, USA. 
Present address: Department of Pathobiology, University of Connecticut, 61 N. Eagleville Road, Storrs, CT 06269, USA.
Present address: Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2522 Vet. Med. Basic Sciences Building, MC-002, 2001 S. Lincoln Avenue, Urbana, IL 61802, USA.
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); outbreaks of this disease occur intermittently in Europe and other parts of the developed world, resulting in highly significant economic losses. CSF is a highly contagious and often fatal haemorrhagic disease of swine, presenting as either an acute or a chronic infection (Paton & Greiser-Wilke, 2003). Disease severity depends on a number of viral and host factors, including age and breed of the pig and virulence of the virus strain (Paton & Greiser-Wilke, 2003).
CSFV is an enveloped virus with a single-stranded, 12.5 kb RNA genome of positive polarity (Rice, 1996). Monocytes and macrophages are the main cell types targeted by CSFV, with infection being non-cytopathic (Knoetig et al., 1999). Replication of CSFV in macrophages is interferon-sensitive; however, viral infection does not lead to interferon induction. Secretion of prostaglandin E (PGE), a downregulator of immune functions, is increased following infection with CSFV, but lymphocyte proliferation is increased (Knoetig et al., 1999). These data suggest that CSFV may manipulate the host-cell response directly or indirectly following infection, to permit efficient viral replication.
Macrophages are important in both the initiation and resolution phases of inflammation (Rauh et al., 2005). Classically activated macrophages, stimulated by gamma interferon (Noel et al., 2004), eradicate invading micro-organisms through direct killing and releasing of pro-inflammatory cytokines, such as interleukin-1 (IL-1) and cytotoxic products such as nitric oxide (NO) (Noel et al., 2004; Rauh et al., 2005). In contrast, alternatively activated macrophages (aaM
), stimulated by cytokines generated by T-helper type 2 (Th2) cells (Noel et al., 2004), promote only the resolution phase of inflammation via induction of angiogenesis, tissue remodelling, wound healing and type II immunity (Rauh et al., 2005). aaM hyporesponsive to pro-inflammatory stimuli resemble tumour-associated macrophages, as well as macrophages from patients with severe and chronic inflammation (Rauh et al., 2005). These cells fail to generate NO from L-arginine and, therefore, do not limit the growth of intracellular pathogens efficiently (Noel et al., 2004).
Activity of NO in cellular defence mechanisms includes participation in tissue injury and the mediation of inflammatory processes and apoptosis (Boucher et al., 1999). NO has also been associated with antiviral activity (Torre et al., 2002). The antiviral properties of NO, including inhibition of viral RNA synthesis, viral protein accumulation and virus release from infected cells, have been reported previously in both DNA and RNA viruses (Pertile et al., 1996; Lin et al., 1997; Xing & Schat, 2000; Torre et al., 2002; Akerstrom et al., 2005; Charnsilpa et al., 2005). Inhibition of the replication of Japanese encephalitis virus, another member of the family Flaviviridae, was correlated with cellular NO production (Lin et al., 1997). Arginase-1 has been shown to be upregulated by DNA viruses (Rogers, 1959; Campadelli-Fiume et al., 1981; Bonina et al., 1984). Here, we have used transcriptional profiling of CSFV-infected macrophages to identify cellular genes whose expression is affected by CSFV infection. Data indicate that CSFV inhibits NO production in macrophages and that suppression of NO in infected cells may be significant for CSFV macrophage host range.
Macrophage cell cultures were prepared from swine peripheral blood mononuclear cells as described by Genovesi et al. (1990). A cDNA microarray, comprising 7712 sequences obtained from swine macrophages, was manufactured as described previously (Afonso et al., 2004). CSFV strain Brescia was obtained from the Animal and Plant Health Inspection Service, Plum Island Animal Disease Center, Orient Point, NY, USA, and was propagated and titrated as described previously (Risatti et al., 2005). Macrophage cell cultures were mock- or CSFV-infected by using an m.o.i. of 10. At 24 h post-infection (p.i.), RNA was isolated, labelled with either Cy3 or Cy5 monoreactive dye and hybridized to the microarray slides as described previously (Afonso et al., 2004). In total, eight slides from three independent experiments were analysed; differential expression measurements based on simultaneous two-colour hybridizations were performed with a GenePix 4000A scanner and GenePix Pro 4.0 software (Axon Instruments). Statistical analysis of the GenePix output was performed by using GeneSpring 7.0 software (Agilent Technologies). Data were normalized and analysed as described previously (Afonso et al., 2004).
Of the host genes examined, approximately 99 % were not affected significantly at 24 h p.i. However, the mRNA levels were increased significantly for 11 genes and reduced significantly for 19 genes (>2.5-fold, P<0.05; Table 1). Genes showing significantly increased mRNA levels included the enzyme arginase-1, interleukin-1 (IL-1), chemokine receptor 4 (CCR4), muscleblind-like 1, chemokine ligand 2, phosphoinositide 3-kinase (PI3K), DAX-1 and four genes of unknown function (Table 1). Genes with significantly reduced expression following CSFV infection were mitochondrial ribosomal protein S25, archain variant 1, angiotensin-converting enzyme, transketolase, osteopontin and 14 other genes of unknown function.
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was done by using Northern blotting as described previously (Afonso et al., 2004), as well as semiquantitative PCR using the SuperScript III One-Step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen). Primers for arginase were 5'-GGCTGGTCTGCTTGAGAAAC-3' and 3'-ATCGCCATACTGTGGTCTCC-5' and for IL-1 were 5'-CAGCCATGGCCATAGTACCT-3' and 3'-CCACGATGACAGACACCATC-5'. Comparison with mock-infected controls confirmed the microarray results, demonstrating increased mRNA levels for these genes during infection (Fig. 1a, b). Arginase activity of mock- or CSFV-infected (m.o.i. of 10) macrophages was determined at 24 h p.i. Cells were washed three times before being lysed. Cell lysates were collected and incubated with L-arginine before being assayed for arginase activity via measuring urea production as described by Corraliza et al. (1994). Colorimetric reaction was measured at 550 nm, using urea as standard for a calibration curve. CSFV-infected macrophage cell cultures showed a significant increase in enzymic activity (Fig. 1c). IL-1 protein expression was confirmed by Western blot as described by Harlow & Lane (1988), using a polyclonal affinity-purified antibody to porcine IL-1/IL-IF2 (R & D Systems) (Fig. 1d).
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). Competition for this substrate by arginase-1 may therefore lead to lower levels of NO production in macrophages (Chang et al., 1998; Gobert et al., 2001; Maarsingh et al., 2006). NO levels of mock- or CSFV-infected (m.o.i. of 10) macrophages were determined at 24 h p.i. Cells were labelled with 4,5-diaminofluorescein (DAF-2) according to Nistri et al. (2002); live cells were examined for fluorescence by using a Leica SP2 confocal microscope. Four random fields containing a mean of 44 cells each from infected and mock-infected cells were recorded at a magnification of x40 using 488 nm excitation. The mean ‘energy’ (fluorescence intensity) and cell size were determined by using Leica software. Data are expressed as the mean energy µm–2 and represented as the mean of three independent experiments. Significantly higher levels of NO were observed in mock-infected cells (Fig. 2a, c). To confirm that the decreased level of NO occurred in virally infected cells, identical cultures were labelled with a 1 : 1000 dilution of mAb WH303 to CSFV structural glycoprotein E2 (Edwards et al., 1991) and anti-mouse secondary antiserum conjugated to Texas red (Kirkegaard and Perry). Immunofluorescence indicated that all cells were infected with CSFV (Fig. 2b). Transcriptional activity of inducible nitric oxide synthase (iNOS), the enzyme that produces NO, was studied by using semiquantitative PCR as described above, using the primers 5'-GACGCCCGGAGCTGTTCCACG-3' and 3'-AGCTGGGTGAACTCCACGCTGG-5'. No changes in mRNA levels were observed for iNOS (Fig. 2d), suggesting that reduced NO levels are caused by increased arginase-1 levels.
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), have suppressed NO production (Rodriguez-Sosa et al., 2002).
The observed reduction in NO, coupled with the increased mRNA levels of arginase-1, PI3K and CCR4 in infected macrophages, resembles a state of macrophage differentiation known as aaM. aaM preferentially recruit Th2 cells through interaction with CCR4 (upregulated 2.5-fold in Table 1
) (Noel et al., 2004), which is a part of the chemokine-receptor system that activates the PI3K pathway (upregulated 2.5-fold in Table 1) (Cronshaw et al., 2004). Furthermore, in vivo, differentiated peritoneal and alveolar macrophages that have positive regulation of PI3K have been characterized as aaM (Rauh et al., 2004). Whilst classically activated macrophages typically induce functions destined to kill viruses, aaM promote the resolution phase leading to the downregulation of inflammation.
Increased gene expression of IL-1 is of interest, as it has been shown to induce NO production in a variety of tissues and acts to suppress apoptosis (Chun et al., 1995; Obermeier et al., 1999). Whilst the increased transcriptional responses of arginase-1 and IL-1 in CSFV-infected macrophages seem contradictory, the induction of arginase-1 may be a virus-induced response aimed to suppress the effects of IL-1. Recent work in murine macrophages infected with Streptococcus pyogenes has supported these findings, with genes encoding both IL-1 and arginase-1 being induced in infected cells, whilst iNOS gene expression remained unchanged (Goldmann et al., 2007). There is also some evidence that the core protein of hepatitis C virus (another member of the family Flaviviridae) may, under certain conditions, inhibit NO production in macrophages or liver tissue (Lee et al., 2001). Recent data suggest that inverse regulation of iNOS and arginase by a host protein phosphatase is possible and important for virus replication (Bonaparte et al., 2006); thus, conceivably, CSFV could affect expression of cellular genes to reduce levels of NO.
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ACKNOWLEDGEMENTS |
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Received 30 March 2007;
accepted 8 July 2007.
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