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Impairment of innate immune responses of airway epithelium by infection with bovine viral diarrhea virus
Veterinary Immunology and Immunopathology 116 (2007) 153–162 www.elsevier.com/locate/vetimm
Impairment of innate immune responses of airway epithelium by infection with bovine viral diarrhea virus M. Al-Haddawi, G.B. Mitchell, M.E. Clark, R.D. Wood, J.L. Caswell * Department of Pathobiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received 31 August 2006; received in revised form 2 November 2006; accepted 19 January 2007
Abstract Bovine viral diarrhea virus (BVDV) infection is an important risk factor for development of shipping fever pneumonia in feedlot cattle, and infects but does not cause morphologic evidence of damage to airway epithelial cells. We hypothesized that BVDV predisposes to bacterial pneumonia by impairing innate immune responses in airway epithelial cells. Primary cultures of bovine tracheal epithelial cells were infected with BVDV for 48 h, then stimulated with LPS for 16 h. Expression of tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) mRNA was measured by quantitative RT-PCR, and lactoferrin concentrations were measured in culture supernatant by ELISA. BVDV infection had no detectable effect on the constitutive expression of TAP and LAP mRNA or lactoferrin concentration in culture supernatant. LPS treatment provoked a significant increase in TAP mRNA expression and lactoferrin concentration in the culture supernatant ( p < 0.01), and these effects were significantly ( p < 0.02, p < 0.01) abrogated by prior infection of the tracheal epithelial cells with the type 2 ncp-BVDV isolate. In contrast, infection with the type 1 ncp-BVDV isolate had no effect on TAP mRNA expression or lactoferrin secretion. LPS treatment induced a significant ( p < 0.001) upregulation of LAP mRNA expression, which was not significantly affected by prior infection with BVDV. These data indicate that infection with a type 2 BVDV isolate inhibits the LPS-induced upregulation of TAP mRNA expression and lactoferrin secretion by tracheal epithelial cells, suggesting a novel mechanism by which this virus abrogates respiratory innate immune responses and predisposes to bacterial pneumonia in cattle. # 2007 Elsevier B.V. All rights reserved. Keywords: Innate immunity; Defensins; Lactoferrin; Bovine viral diarrhea virus; Cattle; Respiratory system
1. Introduction Pneumonic pasteurellosis, caused by Mannheimia haemolytica and other opportunistic pathogens of the family Pasteurellaceae, continues to be the most common cause of morbidity and mortality of feedlot beef cattle (Gagea et al., 2006a). The predisposing influences of viral infection, stresses, and adverse climatic conditions are key to development of
* Corresponding author. Tel.: +1 519 824 4120x54555; fax: +1 519 824 5930. E-mail address: [email protected] (J.L. Caswell). 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.01.007
pulmonary infection with M. haemolytica. Infection with BVDV, a pestivirus in the family Flaviviridae, is a major predisposing cause of pneumonic pasteurellosis in feedlot beef cattle (Martin et al., 1989; Potgieter, 1997; Martin et al., 1999; Fulton et al., 2000; Fulton et al., 2002). Calves experimentally infected with BVDV develop more severe lung lesions following challenge with either M. haemolytica or Histophilus somni, compared to those infected with bacteria alone (Potgieter et al., 1984; Potgieter et al., 1988; Potgieter, 1997). Diagnostic investigations have demonstrated higher prevalences of BVDV infection in calves that die of bacterial pneumonia after arrival in feedlots, compared to those dying of other causes (Shahriar
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et al., 2002; Gagea et al., 2006b). Further, epidemiologic investigations have shown that seroconversion to BVDV is more prevalent in feedlot calves that develop pneumonia than in those that remain healthy, indicating that calves experiencing acute BVDV infections are at greater risk of developing bacterial pneumonia; and that calves with positive or high titres to BVDV on arrival in the feedlot have a lower risk of developing pneumonia (Martin et al., 1989; Martin et al., 1999; O’Connor et al., 2001; Fulton et al., 2002). These findings indicate a predisposing role of this virus in development of pneumonic pasteurellosis, and suggest that the virus may impair the defences that normally protect the lung from bacterial infection. The normal lung is well protected from infection by opportunistic extracellular bacteria through a multilayered system of defences. These defences include entrapment of particles in the airway mucus with subsequent clearance by the ciliated epithelium, recognition and phagocytosis of inhaled particles by resident alveolar macrophages as well as neutrophils and macrophages that are recruited to the lung during the inflammatory response, and a variety of antimicrobial peptides and proteins within the airway and alveolar surface fluid. The latter are produced locally by airway and alveolar epithelial cells and alveolar macrophages, or are derived from serum and enter the lung by specific transport mechanisms or during inflammation (Brogden et al., 2003). These functions can be considered as multiple interacting layers of defence, and it is likely that each must be overcome before a bacterial pathogen reaches the lung and survives to cause pneumonia. Antimicrobial peptides and proteins including defensins and lactoferrin are produced by respiratory epithelial cells and mediate innate defence against microbial pathogens (Diamond et al., 1991; Schonwetter et al., 1995). Their expression is upregulated in bacterial infection (Brock, 2002; Caverly et al., 2003) or following in vitro stimulation with lipopolysaccharide (LPS) or TNF-a (Russell et al., 1996; Diamond et al., 2000b), and cause direct damage to bacterial membranes. However, it is unknown whether and how these antimicrobial defences are impaired by factors that predispose to opportunistic bacterial infection in the lung. BVDV infects macrophages and lymphocytes, and infection of pulmonary alveolar macrophages with ncpBVDV results in depression of phagocytosis, FcR and complement receptor expression, microbicidal activity and secretion of chemotactic factors (Welsh et al., 1995; Potgieter, 1995; Peterhans et al., 2003; Chase et al.,
2004). Acute infection with BVDV causes neutropenia (Wood et al., 2004), and this neutropenia may increase the susceptibility of the lung to bacterial infection. Since BVDV infects airway epithelial cells (Confer et al., 2005; Gagea et al., 2006b), we hypothesize that infection with this virus may impair the mucosal defences of the airways and thus predispose to bacterial pneumonia. The specific objectives of this study were to determine whether non-cytopathic BVDV infection modulates the constitutive and LPS-induced innate immune responses of bovine tracheal epithelial cells, as a potential mechanism by which BVDV infection predisposes to bacterial pneumonia in feedlot cattle. 2. Materials and methods 2.1. Bovine viral diarrhea virus isolates Two isolates of ncp-BVDV were selected based on detection of BVDV antigen in airway epithelium. The ncp-BVDV type 1 was isolated from the ileum of a calf that died with lesions of necrotizing bronchopneumonia, mild enteritis and severe hemorrhagic colitis. The ncp-BVDV type 2 was isolated from the lung of a calf that died of probable bloat, with lung lesions of diffuse pulmonary edema and mild neutrophil infiltrates. Viral stocks of 2nd passage were kindly provided by Dr. S. Carman, Animal Health Laboratory, University of Guelph. Viral titers were assessed using MDBK cells grown in 96-well plates, as previously described (Deregt et al., 1998). 2.2. Tracheal epithelial cell cultures Bovine tracheal epithelial cells were isolated from tracheas collected from slaughter cattle as previously described with modification (Yamaya et al., 1992). Tracheas were obtained 1–3 h after slaughter, and rinsed in transfer solution which consisted of cold sterile phosphate-buffered saline (PBS), 50 mg/ml gentamycin, 300 U/ml penicillin, 300 mg/ml streptomycin and 0.25 mg/ml amphotericin B (Invitrogen Corp, Burlington, ON). The mucosa was dissected, rinsed in transfer solution, and incubated in sterile PBS containing 1% protease (Invitrogen Inc., Catalogue No. 17105–041) at 4 8C overnight then at 37 8C for 15 min. Equine serum was added to a final concentration of 5% to stop the protease action. Epithelial cells were harvested by scraping the mucosa with a sterile scalpel blade, then washed three times by centrifugation at 500 g for 5 min. Cell viability and cell concentration were determined using trypan blue and a hemocytometer.
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24-well culture plates (Corning Inc., Corning, NY, USA) were pre-coated with 1 ml of 3% calf collagen type I (Sigma, Catalogue No. C8919) in sterile PBS, incubated overnight at 37 8C and washed with sterile PBS. Starter medium consisted of 45% Dulbecco’s modified Eagle’s medium (DMEM), 45% Ham’s F-12 (Invitrogen), 10% heat-inactivated horse serum, 0.2 ng/ ml epidermal growth factor, 25 mg/ml bovine pituitary extract, 200 U/ml penicillin, 200 mg/ml streptomycin, 25 mg/ml amphotericin B, 10 mg/ml insulin and 2 mM L-glutamine. The cells were at 106 viable cells/ cm2 in 1ml of starter media, and cultured at 37 8C with 5% CO2 for 2 days. The medium was changed to complete culture medium consisting of DMEM/F12 and Ultroser GTM (serum substitute) (BioSepra, ClergySainte-Christophe, France). Immunohistochemistry was used to confirm that the tracheal epithelium was free of BVDV, as described previously (Haines et al., 1992) using anti-BVDV monoclonal antibody 15C5 cell culture supernatant (Dubovi E, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY). The purity of the cultured tracheal epithelial cells was confirmed by immunocytochemistry. Cytocentrifuge preparations of cultured cells were prepared on charged slides, airdried and fixed in acetone for 15 min. The samples were then processed as described above, using a primary antibody against pancytokeratin (AE1/AE3 M3515, Dako) and vimentin (M0725, Dako). 2.3. Viral infection and LPS treatment of tracheal epithelial cells For each BVDV isolate, the cultured tracheal epithelial cells were treated in triplicate as follows. Subconfluent (50–70%) cell monolayers were washed with sterile PBS, and mock-infected or infected with 6 105.5 of 50% tissue culture-infective dose (TCID50) units of second passage BVDV for 48 h. The supernatant was then removed, 1 ml of fresh complete culture medium with or without 100 ng/ml LPS (Pseudomonas aeruginosa serotype 10, Catalogue No. L8643, Sigma) was added to each well, and the cells were incubated for 16 h (Russell et al., 1996; Diamond et al., 2000a). The supernatant was frozen at 20 8C for later measurement of lactoferrin concentration by enzyme linked immunosorbant assay (ELISA). The susceptibility of tracheal epithelial cells to BVDV infection was determined by immunocytochemistry on cytocentrifuge preparations as described above and by reverse transcriptase PCR (RT-PCR) for BVDV done by the Virology Laboratory, Animal
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Health Laboratory, University of Guelph. Total cellular RNA was harvested from the treated cells for analysis of gene expression. Three replicates of the experiment were conducted using tracheal epithelial cells from different cattle. 2.4. Analysis of TAP and LAP gene expression Total cellular RNA was extracted from cultured tracheal epithelial cells with RNeasy Mini Kit (Qiagen Inc, ON, Canada). Purified RNA was treated with DNase to ensure that there was no contamination with genomic DNA, and quantified using spectrophotometry based on absorbance at 260 nm. Gene expression of TAP and LAP was quantified using the LightCycler (Roche, Mannheim, Germany). 400 ng of RNA was reverse transcribed to cDNA using First Strand cDNA Synthesis Kit with anchor dT primers (Roche, Diagnostic Corporation, USA). PCR amplification was performed using the MasterPlus Sybr Green I kit (Roche Diagnostic Corporation, USA) in the presence of gene-specific forward and reverse primers. The expression of each target gene was determined relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using relative quantitative software (Roche). The consistency of GAPDH mRNA expression was evaluated in all mRNA samples extracted from treated and non-treated cells. Primers for TAP and LAP were designed using Primer 3 and LightCycler Probe Design Software 2.0 (Roche), respectively to amplify 100–200 bp products. Primers for GAPDH were as previously published.(Leutenegger et al., 2000) The primers, product sizes, and NCBI accession numbers were: TAP forward 50 -TCTTCCTGGTCCTGTCTGCT-30 , TAP reverse 50 - GCTGTGTCTTGGCCTTCTTT-30 , 183 bp, NM_174776; LAP forward 50 -AATTCTCAAAGCTGCCGT-30 , LAP reverse 50 -CACAGTTTCTGACTCCGC-30 , 164 bp, NM_203435; GAPDH forward 50 -GGCGTGAACCACGAGAAGTATAA-30 , GAPDH reverse 50 -CCCTCCACGATGCCAAAGT-30 , 120 bp, DQ403066. Gains in fluorescence intensity were assayed at a temperature (57 8C) previously optimized for each gene. Amplification reactions were performed in a final volume of 20 ml, containing 1 ml of each forward and reverse primer (optimal primer final concentration 0.2 mM), 4 ml master mix, 9 ml PCR-grade water, and 5 ml of 1:10 diluted cDNA sample. The heating program was 1 cycle at 95 8C for 15 min followed by 45 quantification cycles consisting of 15 s at 95 8C, 20 seconds at 58 8C and at 72 8C for 25 s. The melting curve analysis was performed by an additional cycle
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consisting of denaturation at 95 8C for 0 s, annealing at 65 8C for 15 s, and melting at 95 8C for 0 s. For each primer set, standard curves were generated to quantify gene expression relative to GAPDH. Standard curves were accepted if the slope fell between 3.2 and 3.4, which corresponded to a reaction efficiency of approximately 2.0. Specific coefficient files were generated from the standard curve for each gene (LightCycler Relative Quantification Software, version 1.0; Roche). Amplicons were purified (QIAquick PCR purification kit; Qiagen), run in 1% agarose gel and sequences determined using an Applied Biosystems 3730 DNA Analyzer (Robarts Research Institute, University of Western Ontario, London, ON, Canada). 2.5. Analysis of lactoferrin expression Lactoferrin concentrations were measured by ELISA in tracheal epithelial cell culture supernatants. Viral infection and LPS stimulation were done in triplicate as described above, except that tracheal epithelial cells were cultured for either 24 or 48 h prior to LPS stimulation. ELISAs were performed in microtiter plates (Immulon 4; Thermo Labsystems, Beverly, MA) coated with 1/100 dilution of polyclonal goat antibovine lactoferrin (Bovine Lactoferrin ELISA Quantitation Kit, Bethyl Lab. Inc., Texas, USA) in 0.05 M carbonate–bicarbonate buffer (pH 9.6), overnight at 4 8C. Plates were washed three times with wash buffer (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0) and blocked with 200 ml of 50 mM Tris (pH 8.0), 0.14 M NaCl, 0.1% BSA for 30 min at room temperature. After removing the blocking buffer and washing three times with wash buffer, a 100 ml volume (diluted 1:10) of sample or lactoferrin standard was added to each well as appropriate, and incubated at 37 8C for 90 min. After washing three times, 100 ml of 105 dilution of goat anti-bovine lactoferrin-horseradish peroxidase-(HRP) conjugate in sample/conjugate buffer [50 mM Tris (pH 8.0), 0.14 M NaCl, 1% BSA, 0.05% Tween 20] was added to each well and incubated for 90 minutes at 37 8C. Wells were again washed repeatedly and 100 ml per well of 3,30 ,5,50 , tetramethylebenzidine (TMB) enzyme substrate (SureBlue Reserve, TMB Microwell peroxidase substrate, KPL, Maryland) solution was added and incubated for 5 to 30 min at room temperature. Reactions were stopped by addition of 100 ml of 2 M H2SO4 to each well, and absorbance was read at 450 nm. All unknowns were assayed in duplicate. A standard curve was generated from lactoferrin standard for each run.
Gene expression of lactoferrin in bovine tracheal epithelial cells was analyzed by RT-PCR using the following primers: forward 50 -TGGATAAAGGGACGCAGAAC-30 , reverse 50 - GGCATTTGAACCACTCAGGT-30 , which amplified a 184 bp product (NCBI accession # AH000852). Amplified RT-PCR products were size-fractionated on a 1% agarose gel, stained with ethidium bromide, and sequenced as above. 2.6. Statistical analysis The general linear mixed model using SAS 9.1.3 (SAS Institute Inc., Cary, NC, USA) was used to compare TAP and LAP mRNA expression between treatment groups, using log transformed data. For analysis of TAP and LAP mRNA expression, F test was used to evaluate overall effects in all 3 experimental replicates, and t test was used to evaluate effects within each replicate. Concentrations of lactoferrin protein were compared using 3-factor multifactorial ANOVA (SAS Institute Inc., Cary, NC, USA). Results are given as means standard error of the mean (S.E.M.) of three experiments using cells from different donor calves unless otherwise indicated. 3. Results 3.1. Purity of bovine tracheal epithelial cells and susceptibility to BVDV infection The primary cell cultures were confirmed as epithelial cells by immunocytochemistry for pancytokeratin. Positive immunoreactivity for pancytokeratin was detected in the cytoplasm of approximately 90% of the cultured cells and no immunoreactivity for vimentin was detected. As expected, infection with either of the 2 ncp-BVDV isolates did not induce cytopathic effect. BVDV immunoreactivity was demonstrated in the cytoplasm of approximately 50% of the epithelial cells at 48 h after exposure to the virus, but not in uninfected cells. BVDV infection of the epithelial cells was confirmed by RT-PCR (data not shown). 3.2. Analysis of TAP and LAP gene expression The expression of GAPDH mRNA was not significantly different in uninfected and BVDV-infected or in untreated and LPS-treated cells. Constitutive (no LPS stimulation) expression of LAP or TAP mRNA was not significantly different in uninfected and infected tracheal epithelial cells, at 3, 6, 12, 24 or 48 h after exposure to either of the two ncp-BVDV isolates (Fig. 1,
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Fig. 1. Expression of TAP mRNA was not significantly different in tracheal epithelial cells infected with type 2 ncp-BVDV compared to uninfected tracheal epithelial cells (in the absence of LPS stimulation). Mean S.E.M. ( p > 0.05, t test).
Fig. 2. LPS treatment induced upregulation of tracheal antimicrobial peptide (TAP) mRNA expression in bovine tracheal epithelial cells. Infection with a noncytopathic type 2 BVDV isolate inhibited the LPSinduced upregulation of TAP mRNA expression. *p < 0.02 vs. uninfected LPS-treated cells, F test. These data correspond to experiment 3 in Table 1.
Table 1). Treatment of tracheal epithelial cells with LPS for 16 h resulted in significant ( p < 0.001), 10- to 48fold upregulation of TAP mRNA expression (Fig. 2). This increase was observed in all 3 replicates, performed on different days using tracheal epithelial cells from different cattle (Table 1). There was variability in LPS-induced TAP mRNA expression by uninfected tracheal epithelial cells taken from the 3 different cattle, both in terms of the magnitude of LPS-
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Fig. 3. LPS treatment induced upregulation of lingual antimicrobial peptide (LAP) mRNA expression in bovine tracheal epithelial cells. Infection with either type 1 or type 2 ncp-BVDV had no significant effect on LAP mRNA expression. ( p > 0.05, F test).
induced TAP expression and the percent upregulation in response to LPS (Table 1). Overall, in the three experimental replicates using tracheal epithelial cells from different animals, the type 2 BVDV isolate significantly ( p < 0.02, F test) impaired the LPS-induced upregulation of TAP mRNA expression; that is, cells infected with type 2 BVDV had lower expression of TAP mRNA compared to uninfected LPS-stimulated cells. In contrast, the effect of the type 1 BVDV isolate on LPS-induced upregulation of TAP mRNA expression was inconsistent and not significant ( p > 0.3) (Fig. 2, Table 1). Expression of LAP mRNA was significantly ( p < 0.001) upregulated 2.1- to 13.5-fold following 16 h of treatment of tracheal epithelial cells with LPS, compared to untreated cells. BVDV infection had no significant effect ( p > 0.05) on LAP mRNA expression in unstimulated or LPS-stimulated cells, in any of the three experiments (Fig. 3). 3.3. Analysis of lactoferrin expression Lactoferrin protein was detected by ELISA in the supernatant of cultured tracheal epithelial cells, but not in culture medium that had not been exposed to tracheal epithelial cells. Expression of lactoferrin mRNA in cultured bovine tracheal epithelial cells was confirmed by RT-PCR, with amplification of a 184 bp product that
Table 1 Relative expression of TAP mRNA (mean S.E.M.) in bovine tracheal epithelial cells infected with type 1 or type 2 ncp-BVDV for 48 h, then treated with LPS for 16 h. The 3 experiments represent replicates using cells isolated from 3 different cattle. Experiment
1 2 3
No BVDV
Type 1 BVDV
Type 2 BVDV
LPS
LPS +
LPS
LPS +
LPS
LPS +
1.04 0.22 0.61 0.03 3.55 0.76
50.52 5.46 20.96 1.55 32.18 1.33
11.59 2.27 1.52 0.49 2.38 0.01
27.49 0.19 16.10 2.27 30.26 0.18
8.69 4.38 1.50 0.019 4.70 0.95
1.92 0.20 10.04 0.13 9.77 1.97
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Fig. 4. LPS treatment resulted in higher concentrations of lactoferrin in the supernatant of primary cultures of bovine tracheal epithelial cells. The lactoferrin concentration was significantly lower in the supernatant of cells infected with a noncytopathic type 2 BVDV isolate for 24 h and stimulated with LPS for 16 h, compare to uninfected cells stimulated with LPS (*p < 0.01 vs. uninfected LPS-treated cells, 3-factor multifactorial ANOVA). In contrast, infection with a type 1 BVDV isolate had no significant effect on lactoferrin concentrations.
was confirmed by sequencing. There was no significant alteration in the levels of lactoferrin in the supernatant of cells infected with either isolate of BVDV for 24 h, compared to that of uninfected cells (data not shown). Treatment of tracheal epithelial cells with LPS for 16 h resulted in a significant ( p < 0.01, 3-factor multifactorial ANOVA) 5.5-fold increase in lactoferrin concentration compared to untreated cells (30.5 3.0 versus 4.4 0.3 ng/ml, respectively) (Fig. 4). Lactoferrin concentrations were significantly lower ( p < 0.01, 3-factor multifactorial ANOVA) in the supernatant of tracheal epithelial cells infected for 24 h with the type 2 isolate of BVD and exposed to LPS, compared to that of uninfected cells exposed to LPS (15.1 4.7 versus 30.5 3.0 ng/ml, respectively) (Fig. 4). In contrast, lactoferrin concentrations were not significantly ( p > 0.05) different in the supernatant
Fig. 5. LPS treatment stimulated higher levels of lactoferrin in the supernatant of primary cultures of bovine tracheal epithelial cells. The method is identical to that shown in Fig. 4, except that cells were stimulated with LPS at 48 h rather than 24 h after BVDV infection. The lactoferrin concentration was significantly lower in the supernatant of type 2 BVDV-infected compared to uninfected cells following stimulated with LPS (*p < 0.01 vs. uninfected LPS-treated cells, 3-factor multifactorial ANOVA).
of cells infected for 24 h with the type 1 isolate of BVDV and treated with LPS, compared to that of uninfected cells exposed to LPS (Fig. 4). In the second experiment, tracheal epithelial cells were infected with BVDV for 48 h instead of 24 h, prior to stimulation with LPS for 16 h. There was no significant alteration in the constitutive levels of lactoferrin in the supernatant of tracheal epithelial cells infected with either isolate of BVDV for 48 h, compared to that of uninfected cells. LPS treatment of uninfected tracheal epithelial cells with LPS resulted in an increase in lactoferrin concentration compared to untreated cells (15.7 1.0 versus 7.1 0.3 ng/ml, respectively), whereas this response was abrogated ( p < 0.01) in the cells infected with type 2 BVDV prior to LPS treatment (BVDV: 10.5 1.0; no BVDV: 7.1 0.3 ng/ ml). The LPS-induced lactoferrin concentrations were similar in the supernatants of type 1 BVDV-infected and uninfected cells. (Fig. 5). Similar results were obtained in an additional replicate of the experiment using a 48hour infection, using tracheal epithelial cells harvested from a different calf (data not shown).
4. Discussion The aim of this study was to investigate a novel mechanism by which BVDV impairs the innate immune responses of airway epithelial cells. The study demonstrated that two ncp-BVDV isolates, type 1 and type 2, were able to infect tracheal epithelial cells in vitro. Quantitative RT-PCR data did not reveal a significant effect of the two ncp-BVDV isolates on the constitutive expression of TAP or LAP mRNA in tracheal epithelial cells. However, the type 2 ncp-BVDV isolate, but not the type 1 isolate, caused significant abrogation of LPSinduced expression of TAP mRNA. Expression of lactoferrin mRNA and secretion of lactoferrin by unstimulated tracheal epithelial cells were detected by RT-PCR and ELISA, respectively. A significant increase in lactoferrin concentration was observed in the supernatant of tracheal epithelial cells exposed to LPS for 16 h. Similar to the results for TAP mRNA expression, a direct effect of type 2 ncp-BVDV on lactoferrin concentrations was not detected, but this virus significantly abrogated the LPS-induced enhancement of lactoferrin production. These findings suggest a novel mechanism whereby viral infections such as BVDV may interfere with innate immune responses of airway epithelial cells, and thereby enhance the susceptibility of the lung to opportunistic bacterial infection.
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BVDV has been shown to infect respiratory epithelial cells in vivo (Confer et al., 2005; Gagea et al., 2006b), but the effect of BVDV infection on the function of these cells has not been previously investigated. In addition to their role as a structural barrier to bacteria, airway epithelial cells contribute to innate pulmonary defense through mucus secretion that prevents colonization of epithelial surfaces, ciliary beating that propels mucus and entrapped particles to the nasopharynx, and secretion of antimicrobial proteins and peptides that are thought to prevent colonization of the mucosa and limit bacterial growth during transit to the nasopharynx. The immunocytochemistry results in this study confirmed that tracheal epithelial cells were readily infected with either of the ncp-BVDV isolates, suggesting a possible effect of the virus on the functions of these cells. Real-time RT-PCR analysis revealed that TAP and LAP mRNA were expressed in cultured bovine tracheal epithelial cells, and that neither ncp-BVDV isolate significantly altered the constitutive level of their expression. b-defensins including bovine TAP and LAP are most recognized for their antimicrobial functions against bacteria, fungi, protozoa and viruses. As cationic peptides, b-defensins are able to interact with pathogens having a negative surface membrane charge (Peschel, 2002). Defensins from rabbit, rat, guinea pig and human have shown direct, rather than cell mediated, in vitro neutralization of herpes simplex virus, vesicular stomatitis and influenza A virus associated with binding of the defensins to the virus (Lehrer et al., 1993). It remains unknown whether BVDV infection has no stimulatory effect on the signaling pathways that lead to TAP and LAP expression, or whether this lack of induction represents an active mechanism of the virus to evade this antiviral response. In support of the latter, in vitro studies have shown that cp-BVDVinduces secretion of type I IFN but ncp-BVDV does not (Adler et al., 1997), and ncp-BVDV infection inhibits endogenous induction of type I IFN induced by other viruses (Nakamura et al., 1995; Charleston et al., 2001; Baigent et al., 2002). Further, b-defensins stimulate acquired immune responses by enhancing the antigen presenting functions of dendritic cells and by activation of memory T cells (Yang et al., 2002). Therefore, inhibition of defensin gene expression might represent a method whereby BVDV evades the antiviral innate and acquired immune responses of the host, although additional investigations are needed to confirm this concept. In the present study, treatment of tracheal epithelial cells with 100 ng/ml LPS for 16 h induced significant 10- to 48-fold upregulation of TAP expression,
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consistent with previous studies where TAP expression was inducible in vivo and in vitro after exposure to bacteria, bacterial products such as LPS, or cytokines such as TNF-a (Russell et al., 1996; Diamond et al., 1996; Diamond et al., 2000a). This inducible response to LPS represents a local antimicrobial response at the epithelial surface (Legrand et al., 1990; Diamond et al., 1996). Although a similar dose of LPS and time of exposure were used in the current study, the upregulation of TAP was higher than what has been reported by other investigators (Diamond et al., 1996). The difference in this response may be due to the degree of cellular differentiation in primary cell cultures, or genotypic differences among different donors. In all 3 replicates of this study, LPS-induced upregulation of TAP mRNA expression was significantly lower in cells infected with the type 2 BVDV isolate than in uninfected cells. In contrast, the type 1 BVDV isolate did not significantly affect the LPS-induced upregulation of TAP mRNA expression. Impaired signaling through the CD14—TLR4—NF-kB pathway is one mechanism by which BVDV might diminish the LPS-induced upregulation of TAP mRNA expression. Suppression of human b-defensin 2 expression and secretion in the nasal epithelium of HIV patients occurs via inhibition of the CD14—TLR4—NF-kB pathway (Bosinger et al., 2004; Alp et al., 2005). Similarly, Bordetella bronchiseptica reduces TAP mRNA expression in the bovine airway epithelium by injecting bacterial proteins into the host cell cytoplasm using a type III secretion system, and this effect is associated with inactivation of the NF-kB pathway (Legarda et al., 2005); and vanadium pentoxide, a component of residual fly ash oil resulting from combustion of crude oil, impairs LPS-induced and IL1b-induced enhancement of TAP mRNA expression (Klein-Patel et al., 2006). Strategies by which viruses interfere with NF-kB signalling include reduced degradation of IkB (inhibitors of NF-kB) by poxviruses, HIV, and Epstein-Barr virus; and impaired translocation of NF-kB to the nucleus during African swine fever virus infection (Santoro et al., 2003). Thus, although the mechanism was not investigated in this study, we speculate that certain strains of BVDV might inhibit induction of TAP mRNA expression by blocking NF-kB activation. Exposure of tracheal epithelial cells to LPS caused upregulation of LAP mRNA expression but at a lower level compared to that of TAP. These results are consistent with other studies, which have shown increased LAP mRNA expression in bovine tracheal epithelial cells after in vitro exposure to LPS or TNF-a (Russell et al., 1996) or associated with acute and
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chronic inflammation in the bovine tongue (Schonwetter et al., 1995). Neither isolate of BVDV affected the constitutive level of LAP gene expression, nor did they affect the LPS-induced upregulation of LAP mRNA expression. The mechanism of induction of LAP mRNA expression has not yet been investigated. Although LAP has shown similarity in gene sequence with TAP (Schonwetter et al., 1995), the effect of BVDV on TAP but not LAP gene expression suggests differential regulation of these two b-defensins. The second objective of this study was to investigate the effect of BVDV on secretion of lactoferrin by tracheal epithelial cells. Lactoferrin is an important antimicrobial protein in airway surface liquid: lactoferrin binds ferric iron and thereby impairs bacterial growth; it quells the immuno-inflammatory response by inactivating LPS and CpG-rich DNA, and also by suppressing expression of adhesion molecules on endothelial cells; and lactoferrin and its cleavage product lactoferricin have direct bactericidal activity and inhibit biofilm formation. Lactoferrin gene expression was both constitutive and inducible by LPS (Teng, 2002; Zheng et al., 2005; Bruckmaier, 2005). In this study, lactoferrin gene expression was detected by RTPCR and lactoferrin protein was detected by ELISA in the supernatant of unstimulated tracheal epithelial cells. These results indicate that lactoferrin is produced and secreted by bovine tracheal surface epithelium, in addition to the serous cells of submucosal glands reported earlier (Inoue et al., 1993). Lactoferrin production by tracheal epithelial cells was stimulated by exposure to LPS. These findings are consistent with previous reports, which showed a rapid increase of lactoferrin concentrations in milk following challenge with bacteria or LPS (Kawai et al., 1999; Hagiwara et al., 2003; Schmitz et al., 2004) or following in vitro exposure of bovine mammary epithelial cells to Staphylococcus aureus or LPS (Wellnitz and Kerr, 2004). In the current study, infection with either isolate of BVDV had no direct effect on the concentration of lactoferrin in the supernatant of tracheal epithelial cells. However, infection of tracheal epithelial cells for 24 or 48 h with the type 2 isolate of BVDV significantly inhibited the LPS-induced increase in the concentration of lactoferrin in the supernatant of these cells. These results indicate that infection with the type 2 ncpBVDV isolate abrogates the LPS-enhanced production of lactoferrin by tracheal epithelial cells, comparable to the effect of this virus on TAP mRNA expression. Information on regulation of lactoferrin gene expression is limited, but a recent study revealed six
NF-kB binding sites in the LPS-responsive regions of the lactoferrin promoter, suggesting that lactoferrin expression is regulated by the NF-kB pathway (Zheng et al., 2005). Thus, it is possible that the effects of BVDV infection on both TAP and lactoferrin expression are mediated by modulation of the CD14—TLR4—NFkB pathway. An alternative mechanism is through regulation of lactoferrin secretion. Soluble or particulate stimuli induce release of specific granules from normal neutrophils, but H1N1 influenza virus inhibits lactoferrin release (Abramson et al., 1984). 5. Conclusion In vitro infection of bovine tracheal epithelial cells with an isolate of ncp-BVDV blocks the LPS-induced upregulation of TAP gene expression and lactoferrin production. Since BVDV is a major contributor to the development of bacterial pneumonia in cattle, the results imply a novel mechanism whereby this virus impairs mucosal innate immune responses and thereby predisposes to bacterial infection of the lung. Acknowledgments We thank Dr. S. Carman, Animal Health Laboratory, University of Guelph for access to archived virus isolates, Barb Jefferson for technical assistance, and William Sears for statistical advice. This work was supported by the Natural Sciences and Engineering Research Council of Canada, the Ontario Ministry of Agriculture and Food Beef Research Program, and the Beef Cattle Research Council of the Canadian Cattleman’s Association. References Abramson, J.S., Parce, J.W., Lewis, J.C., Lyles, D.S., Mills, E.L., Nelson, R.D., Bass, D.A., 1984. Characterization of the effect of influenza virus on polymorphonuclear leukocyte membrane responses. Blood 64, 131–138. Adler, B., Adler, H., Pfister, H., Jungi, T.W., Peterhans, E., 1997. Macrophages infected with cytopathic bovine viral diarrhea virus release a factor(s) capable of priming uninfected macrophages for activation-induced apoptosis. J. Virol. 71, 3255–3258. Alp, S., Skrygan, M., Schlottmann, R., Kreuter, A., Otte, J.M., Schmidt, W.E., Brockmeyer, N.H., Bastian, A., 2005. Expression of beta-defensin 1 and 2 in nasal epithelial cells and alveolar macrophages from HIV-infected patients. Eur. J. Med. Res. 10, 1–6. Baigent, S.J., Zhang, G., Fray, M.D., Flick-Smith, H., Goodbourn, S., McCauley, J.W., 2002. Inhibition of beta interferon transcription by noncytopathogenic bovine viral diarrhea virus is through an interferon regulatory factor 3-dependent mechanism. J. Virol. 76, 8979–8988.
M. Al-Haddawi et al. / Veterinary Immunology and Immunopathology 116 (2007) 153–162 Bosinger, S.E., Hosiawa, K.A., Cameron, M.J., Persad, D., Ran, L., Xu, L., Boulassel, M.R., Parenteau, M., Fournier, J., Rud, E.W., Kelvin, D.J., 2004. Gene expression profiling of host response in models of acute HIV infection. J. Immunol. 173, 6858–6863. Brock, J.H., 2002. The physiology of lactoferrin. Biochem. Cell Biol. 80, 1–6. Brogden, K.A., Ackermann, M., McCray Jr., P.B., Tack, B.F., 2003. Antimicrobial peptides in animals and their role in host defences. Int. J. Antimicrob. Agents 22, 465–478. Bruckmaier, R.M., 2005. Gene expression of factors related to the immune reaction in response to intramammary Escherichia coli lipopolysaccharide challenge. J. Dairy Res. 120–124 72 Spec No. Caverly, J.M., Diamond, G., Gallup, J.M., Brogden, K.A., Dixon, R.A., Ackermann, M.R., 2003. Coordinated expression of tracheal antimicrobial peptide and inflammatory-response elements in the lungs of neonatal calves with acute bacterial pneumonia. Infect. Immun. 71, 2950–2955. Charleston, B., Fray, M.D., Baigent, S., Carr, B.V., Morrison, W.I., 2001. Establishment of persistent infection with non-cytopathic bovine viral diarrhoea virus in cattle is associated with a failure to induce type I interferon. J. Gen. Virol. 82, 1893–1897. Chase, C.C., Elmowalid, G., Yousif, A.A., 2004. The immune response to bovine viral diarrhea virus: a constantly changing picture. Vet. Clin. North Am. Food Anim. Pract. 20, 95–114. Confer, A.W., Fulton, R.W., Step, D.L., Johnson, B.J., Ridpath, J.F., 2005. Viral antigen distribution in the respiratory tract of cattle persistently infected with bovine viral diarrhea virus subtype 2a. Vet. Pathol. 42, 192–199. Deregt, D., Van Rijn, P.A., Wiens, T.Y., van den, H.J., 1998. Monoclonal antibodies to the E2 protein of a new genotype (type 2) of bovine viral diarrhea virus define three antigenic domains involved in neutralization. Virus Res. 57, 171–181. Diamond, G., Kaiser, V., Rhodes, J., Russell, J.P., Bevins, C.L., 2000a. Transcriptional regulation of beta-defensin gene expression in tracheal epithelial cells. Infect. Immun. 68, 113–119. Diamond, G., Legarda, D., Ryan, L.K., 2000b. The innate immune response of the respiratory epithelium. Immunol. Rev. 173, 27–38. Diamond, G., Russell, J.P., Bevins, C.L., 1996. Inducible expression of an antibiotic peptide gene in lipopolysaccharide-challenged tracheal epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 93, 5156–5160. Diamond, G., Zasloff, M., Eck, H., Brasseur, M., Maloy, W.L., Bevins, C.L., 1991. Tracheal antimicrobial peptide, a cysteine-rich peptide from mammalian tracheal mucosa: peptide isolation and cloning of a cDNA. Proc. Natl. Acad. Sci. U.S.A. 88, 3952–3956. Fulton, R.W., Purdy, C.W., Confer, A.W., Saliki, J.T., Loan, R.W., Briggs, R.E., Burge, L.J., 2000. Bovine viral diarrhea viral infections in feeder calves with respiratory disease: interactions with Pasteurella spp., parainfluenza-3 virus, and bovine respiratory syncytial virus. Can. J. Vet. Res. 64, 151–159. Fulton, R.W., Ridpath, J.F., Saliki, J.T., Briggs, R.E., Confer, A.W., Burge, L.J., Purdy, C.W., Loan, R.W., Duff, G.C., Payton, M.E., 2002. Bovine viral diarrhea virus (BVDV) 1b: predominant BVDV subtype in calves with respiratory disease. Can. J. Vet. Res. 66, 181–190. Gagea, M., Bateman, K.G., van Dreumel, T., McEwen, B.J., Carman, S., Archambault, M., Shanahan, R.A., Caswell, J.L., 2006a. Diseases and pathogens causing mortality in Ontario beef feedlots. J. Vet. Diagn. Invest. 18, 8–28. Gagea, M.I., Bateman, K.G., Shanahan, R.A., van Dreumel, T., McEwen, B.J., Carman, S., Archambault, M., Caswell, J.L., 2006b. Naturally occurring Mycoplasma bovis-associated pneu-
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Potgieter, L.N., 1997. Bovine respiratory tract disease caused by bovine viral diarrhea virus. Vet. Clin. North Am. Food Anim. Pract. 13, 471–481. Potgieter, L.N., Helman, R.G., Greene, W., Breider, M.A., Thurber, E.T., Peetz, R.H., 1988. Experimental bovine respiratory tract disease with Haemophilus somnus. Vet. Pathol. 25, 124–130. Potgieter, L.N., McCracken, M.D., Hopkins, F.M., Walker, R.D., Guy, J.S., 1984. Experimental production of bovine respiratory tract disease with bovine viral diarrhea virus. Am. J. Vet. Res. 45, 1582– 1585. Russell, J.P., Diamond, G., Tarver, A.P., Scanlin, T.F., Bevins, C.L., 1996. Coordinate induction of two antibiotic genes in tracheal epithelial cells exposed to the inflammatory mediators lipopolysaccharide and tumor necrosis factor alpha. Infect. Immun. 64, 1565–1568. Santoro, M.G., Rossi, A., Amici, C., 2003. NF-kappaB and virus infection: who controls whom. EMBO J. 22, 2552–2560. Schmitz, S., Pfaffl, M.W., Meyer, H.H., Bruckmaier, R.M., 2004. Short-term changes of mRNA expression of various inflammatory factors and milk proteins in mammary tissue during LPS-induced mastitis. Domest. Anim. Endocrinol. 26, 111–126. Schonwetter, B.S., Stolzenberg, E.D., Zasloff, M.A., 1995. Epithelial antibiotics induced at sites of inflammation. Science 267, 1645– 1648.
Shahriar, F.M., Clark, E.G., Janzen, E., West, K., Wobeser, G., 2002. Coinfection with bovine viral diarrhea virus and Mycoplasma bovis in feedlot cattle with chronic pneumonia. Can. Vet. J. 43, 863–868. Teng, C.T., 2002. Lactoferrin gene expression and regulation: an overview. Biochem. Cell Biol. 80, 7–16. Wellnitz, O., Kerr, D.E., 2004. Cryopreserved bovine mammary cells to model epithelial response to infection. Vet. Immunol. Immunopathol. 101, 191–202. Welsh, M.D., Adair, B.M., Foster, J.C., 1995. Effect of BVD virus infection on alveolar macrophage functions. Vet. Immunol. Immunopathol. 46, 195–210. Wood, R.D., Goens, S.D., Carman, P.S., Deregt, D., Jefferson, B., Jacobs, R.M., 2004. Effect on hematopoietic tissue of experimental infection of calves with noncytopathic type 2 bovine viral diarrhea virus. Can. J. Vet. Res. 68, 42–48. Yamaya, M., Finkbeiner, W.E., Chun, S.Y., Widdicombe, J.H., 1992. Differentiated structure and function of cultures from human tracheal epithelium. Am. J. Physiol. 262, L713–L724. Yang, D., Biragyn, A., Kwak, L.W., Oppenheim, J.J., 2002. Mammalian defensins in immunity: more than just microbicidal. Trends Immunol. 23, 291–296. Zheng, J., Ather, J.L., Sonstegard, T.S., Kerr, D.E., 2005. Characterization of the infection-responsive bovine lactoferrin promoter. Gene 353, 107–117 20–6.
Impairment of innate immune responses of airway epithelium by infection with bovine viral diarrhea virus M. Al-Haddawi, G.B. Mitchell, M.E. Clark, R.D. Wood, J.L. Caswell * Department of Pathobiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received 31 August 2006; received in revised form 2 November 2006; accepted 19 January 2007
Abstract Bovine viral diarrhea virus (BVDV) infection is an important risk factor for development of shipping fever pneumonia in feedlot cattle, and infects but does not cause morphologic evidence of damage to airway epithelial cells. We hypothesized that BVDV predisposes to bacterial pneumonia by impairing innate immune responses in airway epithelial cells. Primary cultures of bovine tracheal epithelial cells were infected with BVDV for 48 h, then stimulated with LPS for 16 h. Expression of tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) mRNA was measured by quantitative RT-PCR, and lactoferrin concentrations were measured in culture supernatant by ELISA. BVDV infection had no detectable effect on the constitutive expression of TAP and LAP mRNA or lactoferrin concentration in culture supernatant. LPS treatment provoked a significant increase in TAP mRNA expression and lactoferrin concentration in the culture supernatant ( p < 0.01), and these effects were significantly ( p < 0.02, p < 0.01) abrogated by prior infection of the tracheal epithelial cells with the type 2 ncp-BVDV isolate. In contrast, infection with the type 1 ncp-BVDV isolate had no effect on TAP mRNA expression or lactoferrin secretion. LPS treatment induced a significant ( p < 0.001) upregulation of LAP mRNA expression, which was not significantly affected by prior infection with BVDV. These data indicate that infection with a type 2 BVDV isolate inhibits the LPS-induced upregulation of TAP mRNA expression and lactoferrin secretion by tracheal epithelial cells, suggesting a novel mechanism by which this virus abrogates respiratory innate immune responses and predisposes to bacterial pneumonia in cattle. # 2007 Elsevier B.V. All rights reserved. Keywords: Innate immunity; Defensins; Lactoferrin; Bovine viral diarrhea virus; Cattle; Respiratory system
1. Introduction Pneumonic pasteurellosis, caused by Mannheimia haemolytica and other opportunistic pathogens of the family Pasteurellaceae, continues to be the most common cause of morbidity and mortality of feedlot beef cattle (Gagea et al., 2006a). The predisposing influences of viral infection, stresses, and adverse climatic conditions are key to development of
* Corresponding author. Tel.: +1 519 824 4120x54555; fax: +1 519 824 5930. E-mail address: [email protected] (J.L. Caswell). 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.01.007
pulmonary infection with M. haemolytica. Infection with BVDV, a pestivirus in the family Flaviviridae, is a major predisposing cause of pneumonic pasteurellosis in feedlot beef cattle (Martin et al., 1989; Potgieter, 1997; Martin et al., 1999; Fulton et al., 2000; Fulton et al., 2002). Calves experimentally infected with BVDV develop more severe lung lesions following challenge with either M. haemolytica or Histophilus somni, compared to those infected with bacteria alone (Potgieter et al., 1984; Potgieter et al., 1988; Potgieter, 1997). Diagnostic investigations have demonstrated higher prevalences of BVDV infection in calves that die of bacterial pneumonia after arrival in feedlots, compared to those dying of other causes (Shahriar
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et al., 2002; Gagea et al., 2006b). Further, epidemiologic investigations have shown that seroconversion to BVDV is more prevalent in feedlot calves that develop pneumonia than in those that remain healthy, indicating that calves experiencing acute BVDV infections are at greater risk of developing bacterial pneumonia; and that calves with positive or high titres to BVDV on arrival in the feedlot have a lower risk of developing pneumonia (Martin et al., 1989; Martin et al., 1999; O’Connor et al., 2001; Fulton et al., 2002). These findings indicate a predisposing role of this virus in development of pneumonic pasteurellosis, and suggest that the virus may impair the defences that normally protect the lung from bacterial infection. The normal lung is well protected from infection by opportunistic extracellular bacteria through a multilayered system of defences. These defences include entrapment of particles in the airway mucus with subsequent clearance by the ciliated epithelium, recognition and phagocytosis of inhaled particles by resident alveolar macrophages as well as neutrophils and macrophages that are recruited to the lung during the inflammatory response, and a variety of antimicrobial peptides and proteins within the airway and alveolar surface fluid. The latter are produced locally by airway and alveolar epithelial cells and alveolar macrophages, or are derived from serum and enter the lung by specific transport mechanisms or during inflammation (Brogden et al., 2003). These functions can be considered as multiple interacting layers of defence, and it is likely that each must be overcome before a bacterial pathogen reaches the lung and survives to cause pneumonia. Antimicrobial peptides and proteins including defensins and lactoferrin are produced by respiratory epithelial cells and mediate innate defence against microbial pathogens (Diamond et al., 1991; Schonwetter et al., 1995). Their expression is upregulated in bacterial infection (Brock, 2002; Caverly et al., 2003) or following in vitro stimulation with lipopolysaccharide (LPS) or TNF-a (Russell et al., 1996; Diamond et al., 2000b), and cause direct damage to bacterial membranes. However, it is unknown whether and how these antimicrobial defences are impaired by factors that predispose to opportunistic bacterial infection in the lung. BVDV infects macrophages and lymphocytes, and infection of pulmonary alveolar macrophages with ncpBVDV results in depression of phagocytosis, FcR and complement receptor expression, microbicidal activity and secretion of chemotactic factors (Welsh et al., 1995; Potgieter, 1995; Peterhans et al., 2003; Chase et al.,
2004). Acute infection with BVDV causes neutropenia (Wood et al., 2004), and this neutropenia may increase the susceptibility of the lung to bacterial infection. Since BVDV infects airway epithelial cells (Confer et al., 2005; Gagea et al., 2006b), we hypothesize that infection with this virus may impair the mucosal defences of the airways and thus predispose to bacterial pneumonia. The specific objectives of this study were to determine whether non-cytopathic BVDV infection modulates the constitutive and LPS-induced innate immune responses of bovine tracheal epithelial cells, as a potential mechanism by which BVDV infection predisposes to bacterial pneumonia in feedlot cattle. 2. Materials and methods 2.1. Bovine viral diarrhea virus isolates Two isolates of ncp-BVDV were selected based on detection of BVDV antigen in airway epithelium. The ncp-BVDV type 1 was isolated from the ileum of a calf that died with lesions of necrotizing bronchopneumonia, mild enteritis and severe hemorrhagic colitis. The ncp-BVDV type 2 was isolated from the lung of a calf that died of probable bloat, with lung lesions of diffuse pulmonary edema and mild neutrophil infiltrates. Viral stocks of 2nd passage were kindly provided by Dr. S. Carman, Animal Health Laboratory, University of Guelph. Viral titers were assessed using MDBK cells grown in 96-well plates, as previously described (Deregt et al., 1998). 2.2. Tracheal epithelial cell cultures Bovine tracheal epithelial cells were isolated from tracheas collected from slaughter cattle as previously described with modification (Yamaya et al., 1992). Tracheas were obtained 1–3 h after slaughter, and rinsed in transfer solution which consisted of cold sterile phosphate-buffered saline (PBS), 50 mg/ml gentamycin, 300 U/ml penicillin, 300 mg/ml streptomycin and 0.25 mg/ml amphotericin B (Invitrogen Corp, Burlington, ON). The mucosa was dissected, rinsed in transfer solution, and incubated in sterile PBS containing 1% protease (Invitrogen Inc., Catalogue No. 17105–041) at 4 8C overnight then at 37 8C for 15 min. Equine serum was added to a final concentration of 5% to stop the protease action. Epithelial cells were harvested by scraping the mucosa with a sterile scalpel blade, then washed three times by centrifugation at 500 g for 5 min. Cell viability and cell concentration were determined using trypan blue and a hemocytometer.
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24-well culture plates (Corning Inc., Corning, NY, USA) were pre-coated with 1 ml of 3% calf collagen type I (Sigma, Catalogue No. C8919) in sterile PBS, incubated overnight at 37 8C and washed with sterile PBS. Starter medium consisted of 45% Dulbecco’s modified Eagle’s medium (DMEM), 45% Ham’s F-12 (Invitrogen), 10% heat-inactivated horse serum, 0.2 ng/ ml epidermal growth factor, 25 mg/ml bovine pituitary extract, 200 U/ml penicillin, 200 mg/ml streptomycin, 25 mg/ml amphotericin B, 10 mg/ml insulin and 2 mM L-glutamine. The cells were at 106 viable cells/ cm2 in 1ml of starter media, and cultured at 37 8C with 5% CO2 for 2 days. The medium was changed to complete culture medium consisting of DMEM/F12 and Ultroser GTM (serum substitute) (BioSepra, ClergySainte-Christophe, France). Immunohistochemistry was used to confirm that the tracheal epithelium was free of BVDV, as described previously (Haines et al., 1992) using anti-BVDV monoclonal antibody 15C5 cell culture supernatant (Dubovi E, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY). The purity of the cultured tracheal epithelial cells was confirmed by immunocytochemistry. Cytocentrifuge preparations of cultured cells were prepared on charged slides, airdried and fixed in acetone for 15 min. The samples were then processed as described above, using a primary antibody against pancytokeratin (AE1/AE3 M3515, Dako) and vimentin (M0725, Dako). 2.3. Viral infection and LPS treatment of tracheal epithelial cells For each BVDV isolate, the cultured tracheal epithelial cells were treated in triplicate as follows. Subconfluent (50–70%) cell monolayers were washed with sterile PBS, and mock-infected or infected with 6 105.5 of 50% tissue culture-infective dose (TCID50) units of second passage BVDV for 48 h. The supernatant was then removed, 1 ml of fresh complete culture medium with or without 100 ng/ml LPS (Pseudomonas aeruginosa serotype 10, Catalogue No. L8643, Sigma) was added to each well, and the cells were incubated for 16 h (Russell et al., 1996; Diamond et al., 2000a). The supernatant was frozen at 20 8C for later measurement of lactoferrin concentration by enzyme linked immunosorbant assay (ELISA). The susceptibility of tracheal epithelial cells to BVDV infection was determined by immunocytochemistry on cytocentrifuge preparations as described above and by reverse transcriptase PCR (RT-PCR) for BVDV done by the Virology Laboratory, Animal
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Health Laboratory, University of Guelph. Total cellular RNA was harvested from the treated cells for analysis of gene expression. Three replicates of the experiment were conducted using tracheal epithelial cells from different cattle. 2.4. Analysis of TAP and LAP gene expression Total cellular RNA was extracted from cultured tracheal epithelial cells with RNeasy Mini Kit (Qiagen Inc, ON, Canada). Purified RNA was treated with DNase to ensure that there was no contamination with genomic DNA, and quantified using spectrophotometry based on absorbance at 260 nm. Gene expression of TAP and LAP was quantified using the LightCycler (Roche, Mannheim, Germany). 400 ng of RNA was reverse transcribed to cDNA using First Strand cDNA Synthesis Kit with anchor dT primers (Roche, Diagnostic Corporation, USA). PCR amplification was performed using the MasterPlus Sybr Green I kit (Roche Diagnostic Corporation, USA) in the presence of gene-specific forward and reverse primers. The expression of each target gene was determined relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using relative quantitative software (Roche). The consistency of GAPDH mRNA expression was evaluated in all mRNA samples extracted from treated and non-treated cells. Primers for TAP and LAP were designed using Primer 3 and LightCycler Probe Design Software 2.0 (Roche), respectively to amplify 100–200 bp products. Primers for GAPDH were as previously published.(Leutenegger et al., 2000) The primers, product sizes, and NCBI accession numbers were: TAP forward 50 -TCTTCCTGGTCCTGTCTGCT-30 , TAP reverse 50 - GCTGTGTCTTGGCCTTCTTT-30 , 183 bp, NM_174776; LAP forward 50 -AATTCTCAAAGCTGCCGT-30 , LAP reverse 50 -CACAGTTTCTGACTCCGC-30 , 164 bp, NM_203435; GAPDH forward 50 -GGCGTGAACCACGAGAAGTATAA-30 , GAPDH reverse 50 -CCCTCCACGATGCCAAAGT-30 , 120 bp, DQ403066. Gains in fluorescence intensity were assayed at a temperature (57 8C) previously optimized for each gene. Amplification reactions were performed in a final volume of 20 ml, containing 1 ml of each forward and reverse primer (optimal primer final concentration 0.2 mM), 4 ml master mix, 9 ml PCR-grade water, and 5 ml of 1:10 diluted cDNA sample. The heating program was 1 cycle at 95 8C for 15 min followed by 45 quantification cycles consisting of 15 s at 95 8C, 20 seconds at 58 8C and at 72 8C for 25 s. The melting curve analysis was performed by an additional cycle
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consisting of denaturation at 95 8C for 0 s, annealing at 65 8C for 15 s, and melting at 95 8C for 0 s. For each primer set, standard curves were generated to quantify gene expression relative to GAPDH. Standard curves were accepted if the slope fell between 3.2 and 3.4, which corresponded to a reaction efficiency of approximately 2.0. Specific coefficient files were generated from the standard curve for each gene (LightCycler Relative Quantification Software, version 1.0; Roche). Amplicons were purified (QIAquick PCR purification kit; Qiagen), run in 1% agarose gel and sequences determined using an Applied Biosystems 3730 DNA Analyzer (Robarts Research Institute, University of Western Ontario, London, ON, Canada). 2.5. Analysis of lactoferrin expression Lactoferrin concentrations were measured by ELISA in tracheal epithelial cell culture supernatants. Viral infection and LPS stimulation were done in triplicate as described above, except that tracheal epithelial cells were cultured for either 24 or 48 h prior to LPS stimulation. ELISAs were performed in microtiter plates (Immulon 4; Thermo Labsystems, Beverly, MA) coated with 1/100 dilution of polyclonal goat antibovine lactoferrin (Bovine Lactoferrin ELISA Quantitation Kit, Bethyl Lab. Inc., Texas, USA) in 0.05 M carbonate–bicarbonate buffer (pH 9.6), overnight at 4 8C. Plates were washed three times with wash buffer (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0) and blocked with 200 ml of 50 mM Tris (pH 8.0), 0.14 M NaCl, 0.1% BSA for 30 min at room temperature. After removing the blocking buffer and washing three times with wash buffer, a 100 ml volume (diluted 1:10) of sample or lactoferrin standard was added to each well as appropriate, and incubated at 37 8C for 90 min. After washing three times, 100 ml of 105 dilution of goat anti-bovine lactoferrin-horseradish peroxidase-(HRP) conjugate in sample/conjugate buffer [50 mM Tris (pH 8.0), 0.14 M NaCl, 1% BSA, 0.05% Tween 20] was added to each well and incubated for 90 minutes at 37 8C. Wells were again washed repeatedly and 100 ml per well of 3,30 ,5,50 , tetramethylebenzidine (TMB) enzyme substrate (SureBlue Reserve, TMB Microwell peroxidase substrate, KPL, Maryland) solution was added and incubated for 5 to 30 min at room temperature. Reactions were stopped by addition of 100 ml of 2 M H2SO4 to each well, and absorbance was read at 450 nm. All unknowns were assayed in duplicate. A standard curve was generated from lactoferrin standard for each run.
Gene expression of lactoferrin in bovine tracheal epithelial cells was analyzed by RT-PCR using the following primers: forward 50 -TGGATAAAGGGACGCAGAAC-30 , reverse 50 - GGCATTTGAACCACTCAGGT-30 , which amplified a 184 bp product (NCBI accession # AH000852). Amplified RT-PCR products were size-fractionated on a 1% agarose gel, stained with ethidium bromide, and sequenced as above. 2.6. Statistical analysis The general linear mixed model using SAS 9.1.3 (SAS Institute Inc., Cary, NC, USA) was used to compare TAP and LAP mRNA expression between treatment groups, using log transformed data. For analysis of TAP and LAP mRNA expression, F test was used to evaluate overall effects in all 3 experimental replicates, and t test was used to evaluate effects within each replicate. Concentrations of lactoferrin protein were compared using 3-factor multifactorial ANOVA (SAS Institute Inc., Cary, NC, USA). Results are given as means standard error of the mean (S.E.M.) of three experiments using cells from different donor calves unless otherwise indicated. 3. Results 3.1. Purity of bovine tracheal epithelial cells and susceptibility to BVDV infection The primary cell cultures were confirmed as epithelial cells by immunocytochemistry for pancytokeratin. Positive immunoreactivity for pancytokeratin was detected in the cytoplasm of approximately 90% of the cultured cells and no immunoreactivity for vimentin was detected. As expected, infection with either of the 2 ncp-BVDV isolates did not induce cytopathic effect. BVDV immunoreactivity was demonstrated in the cytoplasm of approximately 50% of the epithelial cells at 48 h after exposure to the virus, but not in uninfected cells. BVDV infection of the epithelial cells was confirmed by RT-PCR (data not shown). 3.2. Analysis of TAP and LAP gene expression The expression of GAPDH mRNA was not significantly different in uninfected and BVDV-infected or in untreated and LPS-treated cells. Constitutive (no LPS stimulation) expression of LAP or TAP mRNA was not significantly different in uninfected and infected tracheal epithelial cells, at 3, 6, 12, 24 or 48 h after exposure to either of the two ncp-BVDV isolates (Fig. 1,
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Fig. 1. Expression of TAP mRNA was not significantly different in tracheal epithelial cells infected with type 2 ncp-BVDV compared to uninfected tracheal epithelial cells (in the absence of LPS stimulation). Mean S.E.M. ( p > 0.05, t test).
Fig. 2. LPS treatment induced upregulation of tracheal antimicrobial peptide (TAP) mRNA expression in bovine tracheal epithelial cells. Infection with a noncytopathic type 2 BVDV isolate inhibited the LPSinduced upregulation of TAP mRNA expression. *p < 0.02 vs. uninfected LPS-treated cells, F test. These data correspond to experiment 3 in Table 1.
Table 1). Treatment of tracheal epithelial cells with LPS for 16 h resulted in significant ( p < 0.001), 10- to 48fold upregulation of TAP mRNA expression (Fig. 2). This increase was observed in all 3 replicates, performed on different days using tracheal epithelial cells from different cattle (Table 1). There was variability in LPS-induced TAP mRNA expression by uninfected tracheal epithelial cells taken from the 3 different cattle, both in terms of the magnitude of LPS-
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Fig. 3. LPS treatment induced upregulation of lingual antimicrobial peptide (LAP) mRNA expression in bovine tracheal epithelial cells. Infection with either type 1 or type 2 ncp-BVDV had no significant effect on LAP mRNA expression. ( p > 0.05, F test).
induced TAP expression and the percent upregulation in response to LPS (Table 1). Overall, in the three experimental replicates using tracheal epithelial cells from different animals, the type 2 BVDV isolate significantly ( p < 0.02, F test) impaired the LPS-induced upregulation of TAP mRNA expression; that is, cells infected with type 2 BVDV had lower expression of TAP mRNA compared to uninfected LPS-stimulated cells. In contrast, the effect of the type 1 BVDV isolate on LPS-induced upregulation of TAP mRNA expression was inconsistent and not significant ( p > 0.3) (Fig. 2, Table 1). Expression of LAP mRNA was significantly ( p < 0.001) upregulated 2.1- to 13.5-fold following 16 h of treatment of tracheal epithelial cells with LPS, compared to untreated cells. BVDV infection had no significant effect ( p > 0.05) on LAP mRNA expression in unstimulated or LPS-stimulated cells, in any of the three experiments (Fig. 3). 3.3. Analysis of lactoferrin expression Lactoferrin protein was detected by ELISA in the supernatant of cultured tracheal epithelial cells, but not in culture medium that had not been exposed to tracheal epithelial cells. Expression of lactoferrin mRNA in cultured bovine tracheal epithelial cells was confirmed by RT-PCR, with amplification of a 184 bp product that
Table 1 Relative expression of TAP mRNA (mean S.E.M.) in bovine tracheal epithelial cells infected with type 1 or type 2 ncp-BVDV for 48 h, then treated with LPS for 16 h. The 3 experiments represent replicates using cells isolated from 3 different cattle. Experiment
1 2 3
No BVDV
Type 1 BVDV
Type 2 BVDV
LPS
LPS +
LPS
LPS +
LPS
LPS +
1.04 0.22 0.61 0.03 3.55 0.76
50.52 5.46 20.96 1.55 32.18 1.33
11.59 2.27 1.52 0.49 2.38 0.01
27.49 0.19 16.10 2.27 30.26 0.18
8.69 4.38 1.50 0.019 4.70 0.95
1.92 0.20 10.04 0.13 9.77 1.97
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Fig. 4. LPS treatment resulted in higher concentrations of lactoferrin in the supernatant of primary cultures of bovine tracheal epithelial cells. The lactoferrin concentration was significantly lower in the supernatant of cells infected with a noncytopathic type 2 BVDV isolate for 24 h and stimulated with LPS for 16 h, compare to uninfected cells stimulated with LPS (*p < 0.01 vs. uninfected LPS-treated cells, 3-factor multifactorial ANOVA). In contrast, infection with a type 1 BVDV isolate had no significant effect on lactoferrin concentrations.
was confirmed by sequencing. There was no significant alteration in the levels of lactoferrin in the supernatant of cells infected with either isolate of BVDV for 24 h, compared to that of uninfected cells (data not shown). Treatment of tracheal epithelial cells with LPS for 16 h resulted in a significant ( p < 0.01, 3-factor multifactorial ANOVA) 5.5-fold increase in lactoferrin concentration compared to untreated cells (30.5 3.0 versus 4.4 0.3 ng/ml, respectively) (Fig. 4). Lactoferrin concentrations were significantly lower ( p < 0.01, 3-factor multifactorial ANOVA) in the supernatant of tracheal epithelial cells infected for 24 h with the type 2 isolate of BVD and exposed to LPS, compared to that of uninfected cells exposed to LPS (15.1 4.7 versus 30.5 3.0 ng/ml, respectively) (Fig. 4). In contrast, lactoferrin concentrations were not significantly ( p > 0.05) different in the supernatant
Fig. 5. LPS treatment stimulated higher levels of lactoferrin in the supernatant of primary cultures of bovine tracheal epithelial cells. The method is identical to that shown in Fig. 4, except that cells were stimulated with LPS at 48 h rather than 24 h after BVDV infection. The lactoferrin concentration was significantly lower in the supernatant of type 2 BVDV-infected compared to uninfected cells following stimulated with LPS (*p < 0.01 vs. uninfected LPS-treated cells, 3-factor multifactorial ANOVA).
of cells infected for 24 h with the type 1 isolate of BVDV and treated with LPS, compared to that of uninfected cells exposed to LPS (Fig. 4). In the second experiment, tracheal epithelial cells were infected with BVDV for 48 h instead of 24 h, prior to stimulation with LPS for 16 h. There was no significant alteration in the constitutive levels of lactoferrin in the supernatant of tracheal epithelial cells infected with either isolate of BVDV for 48 h, compared to that of uninfected cells. LPS treatment of uninfected tracheal epithelial cells with LPS resulted in an increase in lactoferrin concentration compared to untreated cells (15.7 1.0 versus 7.1 0.3 ng/ml, respectively), whereas this response was abrogated ( p < 0.01) in the cells infected with type 2 BVDV prior to LPS treatment (BVDV: 10.5 1.0; no BVDV: 7.1 0.3 ng/ ml). The LPS-induced lactoferrin concentrations were similar in the supernatants of type 1 BVDV-infected and uninfected cells. (Fig. 5). Similar results were obtained in an additional replicate of the experiment using a 48hour infection, using tracheal epithelial cells harvested from a different calf (data not shown).
4. Discussion The aim of this study was to investigate a novel mechanism by which BVDV impairs the innate immune responses of airway epithelial cells. The study demonstrated that two ncp-BVDV isolates, type 1 and type 2, were able to infect tracheal epithelial cells in vitro. Quantitative RT-PCR data did not reveal a significant effect of the two ncp-BVDV isolates on the constitutive expression of TAP or LAP mRNA in tracheal epithelial cells. However, the type 2 ncp-BVDV isolate, but not the type 1 isolate, caused significant abrogation of LPSinduced expression of TAP mRNA. Expression of lactoferrin mRNA and secretion of lactoferrin by unstimulated tracheal epithelial cells were detected by RT-PCR and ELISA, respectively. A significant increase in lactoferrin concentration was observed in the supernatant of tracheal epithelial cells exposed to LPS for 16 h. Similar to the results for TAP mRNA expression, a direct effect of type 2 ncp-BVDV on lactoferrin concentrations was not detected, but this virus significantly abrogated the LPS-induced enhancement of lactoferrin production. These findings suggest a novel mechanism whereby viral infections such as BVDV may interfere with innate immune responses of airway epithelial cells, and thereby enhance the susceptibility of the lung to opportunistic bacterial infection.
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BVDV has been shown to infect respiratory epithelial cells in vivo (Confer et al., 2005; Gagea et al., 2006b), but the effect of BVDV infection on the function of these cells has not been previously investigated. In addition to their role as a structural barrier to bacteria, airway epithelial cells contribute to innate pulmonary defense through mucus secretion that prevents colonization of epithelial surfaces, ciliary beating that propels mucus and entrapped particles to the nasopharynx, and secretion of antimicrobial proteins and peptides that are thought to prevent colonization of the mucosa and limit bacterial growth during transit to the nasopharynx. The immunocytochemistry results in this study confirmed that tracheal epithelial cells were readily infected with either of the ncp-BVDV isolates, suggesting a possible effect of the virus on the functions of these cells. Real-time RT-PCR analysis revealed that TAP and LAP mRNA were expressed in cultured bovine tracheal epithelial cells, and that neither ncp-BVDV isolate significantly altered the constitutive level of their expression. b-defensins including bovine TAP and LAP are most recognized for their antimicrobial functions against bacteria, fungi, protozoa and viruses. As cationic peptides, b-defensins are able to interact with pathogens having a negative surface membrane charge (Peschel, 2002). Defensins from rabbit, rat, guinea pig and human have shown direct, rather than cell mediated, in vitro neutralization of herpes simplex virus, vesicular stomatitis and influenza A virus associated with binding of the defensins to the virus (Lehrer et al., 1993). It remains unknown whether BVDV infection has no stimulatory effect on the signaling pathways that lead to TAP and LAP expression, or whether this lack of induction represents an active mechanism of the virus to evade this antiviral response. In support of the latter, in vitro studies have shown that cp-BVDVinduces secretion of type I IFN but ncp-BVDV does not (Adler et al., 1997), and ncp-BVDV infection inhibits endogenous induction of type I IFN induced by other viruses (Nakamura et al., 1995; Charleston et al., 2001; Baigent et al., 2002). Further, b-defensins stimulate acquired immune responses by enhancing the antigen presenting functions of dendritic cells and by activation of memory T cells (Yang et al., 2002). Therefore, inhibition of defensin gene expression might represent a method whereby BVDV evades the antiviral innate and acquired immune responses of the host, although additional investigations are needed to confirm this concept. In the present study, treatment of tracheal epithelial cells with 100 ng/ml LPS for 16 h induced significant 10- to 48-fold upregulation of TAP expression,
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consistent with previous studies where TAP expression was inducible in vivo and in vitro after exposure to bacteria, bacterial products such as LPS, or cytokines such as TNF-a (Russell et al., 1996; Diamond et al., 1996; Diamond et al., 2000a). This inducible response to LPS represents a local antimicrobial response at the epithelial surface (Legrand et al., 1990; Diamond et al., 1996). Although a similar dose of LPS and time of exposure were used in the current study, the upregulation of TAP was higher than what has been reported by other investigators (Diamond et al., 1996). The difference in this response may be due to the degree of cellular differentiation in primary cell cultures, or genotypic differences among different donors. In all 3 replicates of this study, LPS-induced upregulation of TAP mRNA expression was significantly lower in cells infected with the type 2 BVDV isolate than in uninfected cells. In contrast, the type 1 BVDV isolate did not significantly affect the LPS-induced upregulation of TAP mRNA expression. Impaired signaling through the CD14—TLR4—NF-kB pathway is one mechanism by which BVDV might diminish the LPS-induced upregulation of TAP mRNA expression. Suppression of human b-defensin 2 expression and secretion in the nasal epithelium of HIV patients occurs via inhibition of the CD14—TLR4—NF-kB pathway (Bosinger et al., 2004; Alp et al., 2005). Similarly, Bordetella bronchiseptica reduces TAP mRNA expression in the bovine airway epithelium by injecting bacterial proteins into the host cell cytoplasm using a type III secretion system, and this effect is associated with inactivation of the NF-kB pathway (Legarda et al., 2005); and vanadium pentoxide, a component of residual fly ash oil resulting from combustion of crude oil, impairs LPS-induced and IL1b-induced enhancement of TAP mRNA expression (Klein-Patel et al., 2006). Strategies by which viruses interfere with NF-kB signalling include reduced degradation of IkB (inhibitors of NF-kB) by poxviruses, HIV, and Epstein-Barr virus; and impaired translocation of NF-kB to the nucleus during African swine fever virus infection (Santoro et al., 2003). Thus, although the mechanism was not investigated in this study, we speculate that certain strains of BVDV might inhibit induction of TAP mRNA expression by blocking NF-kB activation. Exposure of tracheal epithelial cells to LPS caused upregulation of LAP mRNA expression but at a lower level compared to that of TAP. These results are consistent with other studies, which have shown increased LAP mRNA expression in bovine tracheal epithelial cells after in vitro exposure to LPS or TNF-a (Russell et al., 1996) or associated with acute and
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chronic inflammation in the bovine tongue (Schonwetter et al., 1995). Neither isolate of BVDV affected the constitutive level of LAP gene expression, nor did they affect the LPS-induced upregulation of LAP mRNA expression. The mechanism of induction of LAP mRNA expression has not yet been investigated. Although LAP has shown similarity in gene sequence with TAP (Schonwetter et al., 1995), the effect of BVDV on TAP but not LAP gene expression suggests differential regulation of these two b-defensins. The second objective of this study was to investigate the effect of BVDV on secretion of lactoferrin by tracheal epithelial cells. Lactoferrin is an important antimicrobial protein in airway surface liquid: lactoferrin binds ferric iron and thereby impairs bacterial growth; it quells the immuno-inflammatory response by inactivating LPS and CpG-rich DNA, and also by suppressing expression of adhesion molecules on endothelial cells; and lactoferrin and its cleavage product lactoferricin have direct bactericidal activity and inhibit biofilm formation. Lactoferrin gene expression was both constitutive and inducible by LPS (Teng, 2002; Zheng et al., 2005; Bruckmaier, 2005). In this study, lactoferrin gene expression was detected by RTPCR and lactoferrin protein was detected by ELISA in the supernatant of unstimulated tracheal epithelial cells. These results indicate that lactoferrin is produced and secreted by bovine tracheal surface epithelium, in addition to the serous cells of submucosal glands reported earlier (Inoue et al., 1993). Lactoferrin production by tracheal epithelial cells was stimulated by exposure to LPS. These findings are consistent with previous reports, which showed a rapid increase of lactoferrin concentrations in milk following challenge with bacteria or LPS (Kawai et al., 1999; Hagiwara et al., 2003; Schmitz et al., 2004) or following in vitro exposure of bovine mammary epithelial cells to Staphylococcus aureus or LPS (Wellnitz and Kerr, 2004). In the current study, infection with either isolate of BVDV had no direct effect on the concentration of lactoferrin in the supernatant of tracheal epithelial cells. However, infection of tracheal epithelial cells for 24 or 48 h with the type 2 isolate of BVDV significantly inhibited the LPS-induced increase in the concentration of lactoferrin in the supernatant of these cells. These results indicate that infection with the type 2 ncpBVDV isolate abrogates the LPS-enhanced production of lactoferrin by tracheal epithelial cells, comparable to the effect of this virus on TAP mRNA expression. Information on regulation of lactoferrin gene expression is limited, but a recent study revealed six
NF-kB binding sites in the LPS-responsive regions of the lactoferrin promoter, suggesting that lactoferrin expression is regulated by the NF-kB pathway (Zheng et al., 2005). Thus, it is possible that the effects of BVDV infection on both TAP and lactoferrin expression are mediated by modulation of the CD14—TLR4—NFkB pathway. An alternative mechanism is through regulation of lactoferrin secretion. Soluble or particulate stimuli induce release of specific granules from normal neutrophils, but H1N1 influenza virus inhibits lactoferrin release (Abramson et al., 1984). 5. Conclusion In vitro infection of bovine tracheal epithelial cells with an isolate of ncp-BVDV blocks the LPS-induced upregulation of TAP gene expression and lactoferrin production. Since BVDV is a major contributor to the development of bacterial pneumonia in cattle, the results imply a novel mechanism whereby this virus impairs mucosal innate immune responses and thereby predisposes to bacterial infection of the lung. Acknowledgments We thank Dr. S. Carman, Animal Health Laboratory, University of Guelph for access to archived virus isolates, Barb Jefferson for technical assistance, and William Sears for statistical advice. This work was supported by the Natural Sciences and Engineering Research Council of Canada, the Ontario Ministry of Agriculture and Food Beef Research Program, and the Beef Cattle Research Council of the Canadian Cattleman’s Association. References Abramson, J.S., Parce, J.W., Lewis, J.C., Lyles, D.S., Mills, E.L., Nelson, R.D., Bass, D.A., 1984. Characterization of the effect of influenza virus on polymorphonuclear leukocyte membrane responses. Blood 64, 131–138. Adler, B., Adler, H., Pfister, H., Jungi, T.W., Peterhans, E., 1997. Macrophages infected with cytopathic bovine viral diarrhea virus release a factor(s) capable of priming uninfected macrophages for activation-induced apoptosis. J. Virol. 71, 3255–3258. Alp, S., Skrygan, M., Schlottmann, R., Kreuter, A., Otte, J.M., Schmidt, W.E., Brockmeyer, N.H., Bastian, A., 2005. Expression of beta-defensin 1 and 2 in nasal epithelial cells and alveolar macrophages from HIV-infected patients. Eur. J. Med. Res. 10, 1–6. Baigent, S.J., Zhang, G., Fray, M.D., Flick-Smith, H., Goodbourn, S., McCauley, J.W., 2002. Inhibition of beta interferon transcription by noncytopathogenic bovine viral diarrhea virus is through an interferon regulatory factor 3-dependent mechanism. J. Virol. 76, 8979–8988.
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