Reduced expression of psoriasin in human airway cystic fibrosis epithelia

Respiratory Physiology & Neurobiology 183 (2012) 177–185

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Reduced expression of psoriasin in human airway cystic fibrosis epithelia Tiesong Li, Elizabeth A. Cowley ∗ Department of Physiology and Biophysics, Dalhousie University, PO Box 15000, Halifax, Nova Scotia, B3H 2R2, Canada

a r t i c l e

i n f o

Article history: Accepted 24 June 2012 Keywords: Psoriasin Airway epithelia Cystic fibrosis Cytokine Oxidant stress Pseudomonas aeruginosa

a b s t r a c t Psoriasin is a low molecular weight Ca2+ -binding protein with known antimicrobial activity. Since human airway epithelial cells produce a number of powerful antimicrobial agents as part of their host defence, we investigated whether psoriasin was expressed in human bronchial epithelial cell lines. Expression was investigated in 16HBE14o- cells, derived from a normal individual, and compared to CFBE41o- cells, derived from a cystic fibrosis patient. We also examined psoriasin expression following treatment with factors pertinent to the CF lung-oxidant stress and exposure to pro-inflammatory cytokines. CFBE41ocells demonstrated much reduced psoriasin levels compared to the 16HBE14o- cells. Increased psoriasin expression was seen following treatment with IL-22 and a cytomix of the pro-inflammatory cytokines IL-1␤, TNF-␣ and IFN-␥; however, the oxidant stressor tert-butyl hydroperoxide had no apparent effect. Over-expression of human psoriasin into both cell lines resulted in increased internalization of Pseudomonas aeruginosa. In conclusion, expression of psoriasin – which has known anti-microbial activity in other systems – appears to be reduced in CFBE410- compared to 16HBE14o- cells, and its expression modified by exposure to pro-inflammatory cytokines. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Psoriasin (or S100A7) is a low molecular weight, Ca2+ -binding protein originally described in psoriatic skin lesions (Madsen et al., 1991) and involved in keratinocyte differentiation (Broome et al., 2003). Psoriasin possesses powerful antimicrobial activity; for example, it is strongly protective against Escherichia coli in both normal skin (Glaser et al., 2005) and wounds (Lee and Eckert, 2007), and though less potent, does demonstrate antimicrobial activity against S. aureus and Pseudomonas aeruginosa (Glaser et al., 2005). Airway epithelial cells produce a number of powerful antimicrobial agents, often in common with the skin, since both barriers encounter significant amounts of microflora (Hiemstra, 2007; Hata and Gallo, 2008). In the airways, these antimicrobial agents are secreted into the airway surface fluid, where they can act as the first line of defence against inhaled pathogens. Cystic fibrosis (CF) lung disease is characterized by repeated, devastating, cycles of infections with opportunistic pathogens such as P. aeruginosa, many of which are now demonstrating resistance against standard antibiotics. Strategies which would enhance the innate immune system of the CF airway epithelia would potentially be of benefit as an alternate therapeutic approach. We wished to investigate whether airway epithelial cells expressed psoriasin, and hypothesized that

∗ Corresponding author. Tel.: +1 902 494 3805; fax: +1 902 494 1685. E-mail addresses: [email protected] (T. Li), [email protected] (E.A. Cowley). 1569-9048/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.resp.2012.06.028

its expression might be affected by physiological factors pertinent to the CF lung, specifically oxidant stress and exposure to proinflammatory cytokines. Indeed, psoriasin expression is known to be upregulated by exposure to the cytokine interleukin-22 (IL22) in keratinocytes (Wolk et al., 2006), and also oxidant stress in mammary epithelial cells (Carlsson et al., 2005). Thus, we hypothesized that the expression of psoriasin within the airways could play a role in maintaining a sterile environment, while exposure to pro-inflammatory mediators and oxidant stress could result in an enhanced expression representing an adaptive host defence response to combat deleterious bacterial infections. Finally, we wished to determine whether increasing psoriasin expression in model human airway epithelial cells had an effect on the ability of those cell lines to cope with exposure to a mucoid strain of P. aeruginosa, thus suggesting a potentially novel role for this protein in airway epithelial cells. 2. Methods 2.1. Cell culture 16HBE14o- human bronchial epithelial cells and CFBE41o- cystic fibrosis human bronchial epithelial cells (both provided by Dr Dieter Gruenert, California Pacific Medical Centre, San Francisco, CA) were cultured in minimum essential medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 ␮g/ml streptomycin (Invitrogen Life Technologies, Carlsbad, CA). Cells were incubated at 37 ◦ C in humidified 5% CO2 /95% air. For RNA and

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protein extraction, cells were cultured on 100 mm diameter Falcon culture dishes (Becton Dickinson, Franklin Lanes, NJ). For cell treatments, regular culture media was removed and replaced with one containing no FBS, either in the presence of the agent of interest or a vehicle control, for 24 h. 2.2. RNA extraction, RT-PCR and quantitative PCR Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse transcription performed using MMLV reverse transcriptase (Invitrogen) in the presence of 5 mM dNTP (Invitrogen) and 1 ␮M oligo dT (Amersham Pharmacia, Baie D’Urfe, PQ, Canada). PCR was performed to investigate psoriasin and IL-22 receptor (IL-22RA1 and IL-10RB) mRNA expression using the following primers (forward, reverse): psoriasin: 5 -CCAGCAAGGACAGAAACT-3 and 5 -AAGCAAAGATGAGCAACAC-3 ; IL-10RB: 5 GTCAGGGCTGAATTTGCA-3 and 5 -CCCTCGAACTTGAACACA-3 ; IL-22RA1: 5 -CTACTATGCCAGGGTCA-3 and 5  CTCTGTGTCAGGGGTCA-3 . Primers (Invitrogen) were used at 400 nM. PCR experiments were performed in the presence of 1.5 mM MgCl2 , 10X Taq buffer with KCl, 2.5 U Taq polymerase (all from MBI Fermentas, Burlington, ON, Canada) and 5 mM dNTP (Invitrogen) in a total reaction volume of 25 ␮l. After an initial denaturation at 95 ◦ C for 1 min, the samples were denatured at 94 ◦ C for 30 s, allowed to anneal at 52 ◦ C (55 ◦ C for IL-10RB and IL-22RA1) for 30 s, and extended at 72 ◦ C for 30 s for 35 cycles. Reactions were completed by a final extension at 72 ◦ C for 10 min. Each PCR reaction was performed at least 3 times on different passages of cells. Quantitative PCR (qPCR) was used to investigate changes in psoriasin gene expression following exposure to our treatments of interest when compared to that of the housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT), using the Lightcycler thermal cycler system (Roche Applied Science, Laval, PQ, Canada) as described in Roy et al., 2006. Primers sequences were 5 -GCCAGACTTTGTTGGATTTG-3 and 5 CTCTCATCTTAGGCTTTGTATTTTG-3 , with conditions as follows: 10 min at 95 ◦ C, followed by 45 cycles of 95 ◦ C for 10 s, 60 ◦ C (for HPRT) or 52 ◦ C (for psoriasin) for 5 s and 72 ◦ C for 10 s. The amount of psoriasin transcript was normalized to the level of HPRT transcript (which was unaffected by any of the treatments) and normalized data subjected to analysis of variance (ANOVA) followed by a Tukey’s post hoc test as appropriate. All values are reported as mean ± S.E.M. with statistical significance reported at p < 0.05. 2.3. Immunoblotting Cells were scraped, spun down and the pellet resuspended in a lysis buffer containing 10% SDS and 15 mg/ml DTT in the presence of HaltTM protease inhibitor (Thermo Scientific, Ottawa, ON, Canada). Total protein lysate was run on a 12.5% polyacrylamide gel and transferred to nitrocellulose membrane (Bio-Rad Laboratories, Mississauga, ON, Canada). Immunoblotting to confirm psoriasin protein expression was performed using a mouse anti-human psoriasin monoclonal antibody (clone # 47C1068; Imgenex, San Diego, CA) at 1:750 dilution. After incubation with the primary antibody, the membrane was incubated with a goat-anti-mouse horse-radish peroxidase (HRP)-conjugated secondary antibody in 5% non-fat milk at 1:7500 dilution (all Jackson ImmunoResearch, West Grove, PA). Proteins were detected using an ECL Plus kit (Amersham Pharmacia). Unfortunately, peptides corresponding to the epitope sequences of the psoriasin antibody were not commercially available and thus could not be used to confirm specificity. Therefore, control experiments consisted of those in which the primary antibody was omitted (results not shown). Additionally, protein lysate

from the human mammary epithelial cell line MCF-10A was used as a positive control (Carlsson et al., 2005). For protein quantification, a densometric analysis was performed by scanning the film using a UMAX Powerbook III (UMA, Dallas, TX) and the band images quantified using ImageJ software (version 1.39, National Institute of Health) using ␤-actin as the housekeeping protein. The ␤-actin primary antibody (rabbit antihuman polyclonal #4967; Cell Signalling Technology, Danvers, MA) was used at 1:1000 dilution, while an HRP-conjugated goat antirabbit IgG (Jackson ImmunoResearch) was used at 1:5000 dilution as the secondary.

2.4. MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) assay Cell viability was determined using the colorimetric MTT assay, which is based on the production of MTT-formazan crystals from MTT via the activity of mitochondrial enzymes. Briefly, cells were plated on a 96-well plate and treated with tert-butylhydroperoxide for 24 h. After this, MTT (5 mg/ml; Sigma–Aldrich, Oakville, Ontario, Canada) was added to each well. Following incubation at 37 ◦ C for 4 h, the produced formazan product was extracted with acid–isopropanol solution (0.04 N HCl/isopropanol) and the absorbance at 570 nm was determined using a Beckman Coulter AD340 microplate reader (Beckman Coulter, Inc., Fullerton, Ontario, Canada). Results were determined in relation to healthy, untreated control cells.

2.5. P. aeruginosa internalization assay and psoriasin transfection We wished to investigate whether over-expression of psoriasin in these cell lines had any effect on their ability to cope with infection by a clinically relevant mucoid P. aeruginosa strain utilizing a bacterial internalization and antibiotic protection assay. P. aeruginosa strain 8821 (a gift from Dr T.J. Lin, Dalhousie University), a mucoid strain isolated from a cystic fibrosis patient (Kamath et al., 1998) were cultured in Luria–Bertani broth (10 g/L NaCl, 10 g/L Bacto-Tryptone, 5 g/L Yeast Extract) overnight to reach an O.D. at 620 nm of 2.5–3.0 U (1 × 109 bacteria/U, early stationary phase). Bacteria were prepared as previously described by Power et al. (2007). Initially both cell lines were inoculated with P. aeruginosa corresponding to a multiplicity of infection (MOI) of 25 bacteria per cell in order to investigate any innate difference between the two cell lines to cope with infection. After 90 min of exposure to P. aeruginosa, an antibiotic mixture (50 U/ml penicillin, 50 U/ml streptomycin, 200 ␮g/ml gentamicin (Invitrogen), 100 ␮g/ml ceftazidine (Sigma–Aldrich) and 100 ␮g/ml piperacillin (Pharmaceutical Partners of Canada, Richmond Hill, Ontario, Canada) was added for another 60 min to kill any remaining extracellular bacteria. After this, the cells were lysed with 0.1% Triton-X to release intracellular bacteria. This lysate was diluted 1:2 with PBS, plated on agar and grown overnight prior to the colonies being counted. For the transfection experiments, both 16HBE14o- and CFBE41o- cells were transfected when they were at approximately 50% confluence with either psoriasin-pCMV6-XL5 (OriGene Technologies, Inc.) or empty vector control (pCMV6-XL5) in OPTI-MEM I solution (Invitrogen) mixed with the transfection reagent MegaTran 1.0 (OriGene) at a ratio of 3:1 MegaTran:DNA. Cells were then incubated at 37 ◦ C for either 48 or 72 h. After this time, transfected cells were washed three times in serum-free media before P. aeruginosa was added as above.

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Fig. 1. Psoriasin expression in human airway epithelial cells. A. cDNA transcripts were detected for psoriasin (246 bp) in both 16HBE14o- and CFBE41o- cells M = 100 bp marker. B. Using qPCR, the relative expression of psoriasin transcript (expressed as total number of copies/␮l RNA) was considerably less in the CFBE41o- cells than the 16HBE14o- (n = 9). C. Immunoblotting of protein from total cell lysate from HBE and CFBE cells demonstrated the presence of psoriasin protein, though expression was extremely low in CFBE cells. +ve is a sample of lysate from MCF-10A mammary epithelial cells, known to express high levels of psoriasin protein.

2.6. Materials Human recombinant IL-1␤, TNF-␣, IFN-␥ and IL-22 were purchased from R&D Systems (Minneapolis, MN) and tert-butyl hydroperoxide (t-BOOH) from Sigma–Aldrich. 3. Results 3.1. Expression of psoriasin in model human airway epithelial cells Psoriasin mRNA expression was confirmed via RT-PCR in 16HBE14o- and CFBE41o- cells (Fig. 1A). The fragment detected was of the predicted size (246 bp), and was not detected without reverse transcription or when water was substituted for the template (results not shown). Using qPCR, we next examined the relative expression of psoriasin transcript between the two cell lines as total number of copies per ␮l RNA. Using this approach, we determined that CFBE41o- cells express significantly less psoriasin mRNA than 16HBE14o- (Fig. 1B). Protein expression was confirmed via immunoblotting on total cell lysate from the two cell lines (Fig. 1C). The protein detected was of the correct size (11.4 kD). Although the protein was detected, it was apparent that the CFBE41o- cells expressed considerably less psoriasin than 16HBE14o- cells, since equivalent amounts of total protein were loaded (30 ␮g). 3.2. Psoriasin expression is unchanged in response to oxidant stress We next investigated whether exposure to oxidant stress affected psoriasin expression in these airway epithelial cell lines, as has previously been reported for mammary epithelia (Carlsson et al., 2005). Replicating the protocol of Carlsson et al. (2005),

we used hydrogen peroxide (H2 O2 , 100 ␮M) for 1 h as the oxidant stressor, and cells were then allowed to recover for two days. However, in neither cell line did this protocol result in any significant changes in psoriasin levels as measured by qPCR (results not shown). Therefore we decided next to investigate whether a longer exposure to oxidant stress, which is highly pertinent to the CF airways, affected psoriasin expression. To do this, we utilized the more stable oxidant stressor t-BOOH, widely used to induce oxidant stress (Jung et al., 1998; Matsuno et al., 2008). In order to establish a non-cytotoxic dose of t-BOOH, we exposed cells to increasing concentrations of t-BOOH and monitored cell viability using an MTT assay. After 24 h, no significant cell death was apparent in the 16HBE14o- cells at t-BOOH concentrations between 50 and 500 ␮M, while 1000 ␮M was toxic to nearly all of the cells (Fig. 2A). In contrast, the CFBE41o- cells succumbed to t-BOOH at much lower concentrations (Fig. 2B); for example, application of 200 ␮M tBOOH resulted in a cell viability of 98.9 ± 2.1% for the 16HBE14ocells versus 4.2 ± 1.3% for the CFBE41o- cells, suggesting the CF cells are inherently less able to deal with exposure to oxidant stress. We next examined psoriasin protein expression over a range of different concentrations of t-BOOH. 16HBE14o- cells were treated with between 0 and 500 ␮M t-BOOH for 24 h, protein extracted and immunoblotting performed. Concentrations greater than 500 ␮M t-BOOH induced so much cell death that it was not possible to extract protein. Following normalization to the house-keeping protein ␤-actin, there was no change in psoriasin expression with oxidant stress (Fig. 2C and D). A similar pattern was apparent with the CFBE41o- cells, which were treated with between 0 and 100 ␮M t-BOOH. Again, higher concentrations did not permit protein extraction due to the high levels of cell death. In this case, the level of protein expression was so low (Fig. 2C) that it was impossible to perform quantification; however, there is no change in signal by eye. This lack of effect on psoriasin expression is in contrast to

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Fig. 2. Effects of the oxidant stressor tert-butylhydroperoxide (t-BOOH) on cell viability and psoriasin expression. A. Application of increasing doses of t-BOOH to 16HBE14ocells had little effect on cell viability prior to 1000 ␮M, as determined by MTT assay. In contrast, CFBE41o- cell succumbed at a much lower dose (B). Results are expressed as the mean ± s.e.m. of 4 individual experiments, with 8 replicates/dose. Panel C shows immunoblotting for psoriasin and ␤-actin protein from cells treated with increasing doses of t-BOOH. Densometric analysis was possible only for the 16HBE14o- cells, but revealed no difference in psoriasin expression with increasing oxidant stress (D). *Significance at p < 0.05 as determined by ANOVA, followed by Tukey’s post hoc test.

the results observed in mammary cells, where protein expression was increased (Carlsson et al., 2005) and suggests differential roles for this protein between the different cell types. 3.3. Effects on psoriasin expression following IL-22 treatment Interleukin (IL)-22 has been proposed as an agent that increases the innate immunity of tissues (Whittington et al., 2004; Wolk et al., 2004) and which mainly targets epithelial cells, including those of the respiratory and gastrointestinal systems (Wolk et al., 2004). It has also been reported to upregulate psoriasin expression in keratinocytes (Wolk et al., 2006); thus we wished to investigate whether it affected psoriasin expression in airway epithelia. IL-22 achieves its effects through a receptor composed of two subunits – the IL-22RA1 and IL-10RB chains – which co-assemble to produce a functional IL-22R complex (Kotenko et al., 2001). To the best of our knowledge there have been no previous reports concerning the expression of the IL-22R complex in these cell lines; therefore we first examined whether the IL-22RA1 and IL-10RB subunits were present and thus able to form a functional receptor complex. Indeed, we were able to detect mRNA expression for both subunits in both cell lines via RT-PCR (Fig. 3A). Next we investigated psoriasin gene expression in response to 24 h of IL-22 treatment. 16HBE14o- cells treated for 24 h

demonstrated a significant increase in psorasin gene expression: treatment with 40 ng/ml increased expression to 160 ± 3.9% compared to untreated cells (Fig. 3B), while 100 ng/ml resulted in an increase to 242 ± 14.5% (results not shown). Cell viability after treatment with IL-22 was measured via the MTT assay, with no significant cell death apparent at either 40 ng/ml or 100 ng/ml for either cell line. We then treated CFBE41o- cells with the lower dose of IL-22 to see whether there was any effect on psoriasin expression; however, no change was apparent (Fig. 3B). Finally, we investigated whether IL-22 treatment had any effect on psoriasin protein expression. Treatment of 16HBE14o- cells with 40 ng/ml or 100 ng/ml IL-22 resulted in modest, but significant, increases in psoriasin protein of 184.8 ± 19.3% and 148.5 ± 6%, respectively (as normalized to ␤-actin expression). The same low, almost non-detectable, level of protein was observed in the CFBE41o- cells precluding quantification (Fig. 3C and D). 3.4. Effects of exposure to pro-inflammatory cytokines on psoriasin expression Although there are no previous reports of psoriasin expression being affected by exposure to pro-inflammatory cytokines, since high levels of pro-inflammatory cytokines have been measured in the CF airway (Bonfield et al., 1999), we wished to investigate

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that had been internalized during the course of the initial 90 min exposure) in the CFBE41o- cells when normalized to controls (Fig. 5A). Next, we employed a transient transfection model to increase psoriasin expression in both cell lines using a commercially available human psoriasin construct. After 48 h transfection, there was an increased psoriasin expression of approximately two-fold in 16HBE14o- cells compared to non-transfected and empty-vector control cells (Fig. 5B), while the fold-increase in the CFBE41o- cells was somewhat higher (approximately fourfold) over the non-transfected controls. The protein level in the psoriasin-transfected CFBE41o- cells was higher than in the nontransfected 16HBE14o- cells (167.5 ± 6.2% vs 100%; n = 6; Fig. 5B and C). Cells (16HBE14o- and CFBE41o-) transfected with psoriasin for 48 and 72 h were then exposed to P. aeruginosa for 90 min (as above). Again, the numbers of intracellular, internalized bacteria were counted to investigate whether increasing psoriasin protein expression affected the numbers of bacteria internalized during this short time-frame of infection. In both cell lines the numbers of intracellular P. aeruginosa were significantly reduced following psoriasin transfection compared to the empty vector control. Due to high inter-experimental variability, internalized P. aeruginosa numbers from psoriasin-transfected cells were normalized to the numbers from vector-alone controls when equivalent bacterial numbers were applied. In 16HBE14o- cells, intracellular bacterial counts were 69.7 ± 5.4% and 68.7 ± 8.7% of control after 48 and 72 h, respectively of psoriasin transfection (both n = 3; Fig. 5D), while for the CFBE41o- cells, levels fell to 63.1 ± 3.7% (n = 5) and 64.7 ± 4.8% (n = 4) after 48 and 72 h (Fig. 5E).

4. Discussion

Fig. 3. Effects of IL-22 treatment on psoriasin expression. RT-PCR confirmed the expression of two subunits (IL-22RA1 and IL-10RB chains) which co-assemble to produce a functional IL-22R complex in both cell types (A). Treatment of cells with IL-22 (40 ng/ml, 24 h) increased psoriasin mRNA expression in 16HBE14o- cells, but had no effect on CFBE41o- cells. Data are normalized to untreated 16HBEo- values. (B). This increase was also apparent via western blotting (Panels C and D). Psoriasin protein was barely detectable in the CFBE41o- cells. Whole cell lysate from MCF-10A cells is shown as a positive control.

whether exposure to IL-1␤, TNF-␣ or IFN-␥ would have any effect on psoriasin expression. Both gene and protein expression were investigated via qPCR and immunoblotting. Exposure to IL-1␤ (2.5 ng/ml) for 24 h had no effect on psoriasin gene expression in the 16HBE14o- cells; however, a significant increase was seen at the protein level (Fig. 4A and B). A significant increase was also seen at the gene level in the CFBE41o- cells (Fig. 4A); however, psoriasin protein expression was again too low to permit accurate quantification. Treatment with TNF-␣ or IFN-␥ (both at 100 ng/ml for 24 h) resulted in significant increases at the gene level in both cells lines. However, this was not seen at the protein level in 16HBE14o- cells (Fig. 4C–F). Finally, treatment with a cytomix of IL-1␤, TNF-␣ and IFN-␥ together resulted in increases in psoriasin gene and protein expression (Fig. 4F and G). 3.5. Increased expression of psoriasin enhances P. aeruginosa internalization Initially, P. aeruginosa strain 8821 was applied to both cell lines for 90 min prior to the application of an antibiotic combination. When the cells were then lysed, permitting the intracellular bacteria to be plated and quantified, there was a statistically significant increase in the number of intracellular P. aeruginosa (i.e. bacteria

We here demonstrate psoriasin mRNA and protein expression in a human airway epithelial cell line widely used as a model of normal bronchial epithelium (16HBE14o-) and in a model CF cell line (CFBE41o-), in which expression was severely reduced. Psoriasin expression has previously been described in tissue taken from large cell lung carcinomas (Zhang et al., 2008); however, we believe this is the first report of this protein in cell lines derived from normal, noncancerous, airway epithelia. The cell lines used in this study were not derived originally from cancerous sources and are considered to reflect a normal, and CF, phenotype respectively (Cozens et al., 1994; Kunzelmann et al., 1993; Ehrhardt et al., 2002). Although widely used as model cell lines, it is important to note that they are not a matched pair of CF versus non-CF cell lines; furthermore, our findings relate only to these particular cell lines, and that caution must be used when extrapolating these findings to normal and CF airway phenotypes. Psoriasin reportedly plays a number of important and diverse biological roles throughout the body, though its potential role in the airways is only beginning to be investigated. Our initial finding was that CFBE41o- cells expressed severely reduced levels of psoriasin mRNA and protein when compared to the 16HBE14ocells. This suggests to us that whatever the biological role of psoriasin in 16HBE14o- airway epithelial cells, this role will be severely compromised in the CFBE41o- cells. Thus, we sought to investigate whether its expression could be modified under conditions of physiological stress pertinent to airway infection and inflammation, hypothesizing that such changes may suggest a potential role for psoriasin in the airways. To do this, we investigated whether a number of agents affected psoriasin expression based either on (a) their known ability to regulate psoriasin in other systems or (b) their potential relevance to CF, and inflammatory lung disease in general. IL-22, a cytokine reported to increase psoriasin expression in skin cells (Wolk et al., 2006), also had the same effect on the

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Fig. 4. Effects of cytokine exposure on psoriasin expression. qPCR and immunoblotting revealed differential changes in expression following treatment of 16HBE14o- and CFBE41o- cells with IL-1␤ (2.5 ng/ml; A and B), TNF-␣ (100 ng/ml; C and D), IFN-␥ (100 ng/ml; E and F) or a cytomix containing all three (G and H), all for 24 h. For the qPCR experiments, data are expressed in relation to the control 16HBEo- samples, which are set at 100%. Densometric analysis was attempted by also probing membranes for ␤-actin; however, the levels of psoriasin protein were so low that accurate quantification was not possible. *Significance at p < 0.05 as determined by ANOVA, followed by Tukey’s post hoc test.

16HBE14o- cells at both the gene and protein levels, while there was no apparent effect on the CFBE41o- cells (Fig. 3). This lack of an effect with the CFBE41o- cells may be a reflection of our finding that mRNA levels are so low in this cell line, it is simply below the level of sensitivity of our qPCR approach to detect changes, or it may reflect another phenotypic difference between the cell lines. During the course of this study we do clearly identify expression of IL-22 receptor components in both these airway epithelial cell lines, which have not been previously described, while reporting that IL-22 application has at least one significant potential biological effect on 16HBEo- cells (i.e. up-regulation of psoriasin expression). Although it has not been studied extensively in the respiratory system, there is an increasing body of evidence that

IL-22 plays several important roles in pulmonary epithelial biology. For example, it appears that IL-22 plays a crucial role in mediating pulmonary host defence against Gram-negative bacteria in a model of murine pneumonia, partly via increasing production of antimicrobial peptides (Aujla et al., 2008; Aujla and Kolls, 2009). It also ameliorates airway inflammation and tissue damage in a murine-model of allergic lung disease (Taube et al., 2011), while Hoegl et al. (2011) recently reported that inhaled IL-22 protects against ventilator-induced lung injury in a rat model. These authors also demonstrated expression of a functional IL-22 receptor in the model human alveolar epithelial cell line, A549. We here demonstrate for the first time expression of the components of a functional IL-22 receptor in the model cell lines 16HBE14o- and CFBE41o-. Thus, we believe these cells lines may represent useful in vitro

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Fig. 5. Transient over-expression of psoriasin increases P. aeruginosa internalization in airway epithelial cells. P. aeruginosa strain 8821 was added to 16HBEo- and CFBE41ocells at an MOI of 22 for 90 min, after which antibiotics were added to kill external bacteria. Significantly more bacteria were internalized in the CFBE41o- cells compared to 16HBEo-. Bacterial colonies were counted in triplicate on 6 separate occasions. Human psoriasin (propagated in pCMV6-XL5) was transfected into 16HBE14o- and CFBE41ocells for either 48 or 72 h. Panel B shows a representative western blot using antibodies against psoriasin and ␤-actin for non-transfected, empty vector controls and psoriasintransfected cells after 48 h transfection. Densometric analysis (Panel C) reveals the relative expression of psoriasin protein compared to 16HBEo- controls (n = 3). Following psoriasin transfection, there is a significant decrease in the number of intracellular P. aeruginosa seen compared to empty vector controls seen for both cell lines after either 48 or 72 h of transfection. Bacterial colonies were counted in quadruplicate on between 3 and 5 separate occasions. *Significance at p < 0.05 vs the appropriate empty vector control as determined by Student’s t-test.

models to investigate the mechanism of IL-22 action on airway epithelia, which would appear to be an area of growing interest. Modest, but significant, increases in psoriasin mRNA and protein were also detected in response to a cytomix of pro-inflammatory cytokines (Fig. 4) suggesting a possible role for this product in an adaptive host defence response against inflammatory mediators. Caution must again be applied in interpreting the results in the CFBE41o- cells, since the copies numbers are so low. However, statistically significant increases in psoriasin mRNA were seen in 16HBE14o- cells in response to IL-1␤, TNF-␣ and IFN-␥ individually as well as together (Fig. 4). However, differences apparent in the 16HBEo- cells at the gene level were not always seen with protein, and vice versa, possibly as a result of the low number of experimental trials (three) for each data point. However, there was a clear and unequivocal increase in psoriasin message and protein in response to the cytomix, clearly demonstrating that exposure to pro-inflammatory cytokines can affect expression. However, it is impossible to say which is the key cytokine triggering this increase or indeed, whether the combination of all three cytokines is required. These are the first reports of the effects of these important inflammatory mediators on psoriasin expression using model airway epithelial cells. Psoriasin has been reported to be

down-regulated in response to IFN-␥ in human breast cancer epithelial cell lines (Petersson et al., 2007), though the apparent discrepancy between our results and those in breast cancer cells may be related to differences in downstream signaling targets in the cell types investigated. We were keen to investigate the role of exposure to oxidant stress, since this is known to play a central role in the pathogenesis and progression of a number of inflammatory pulmonary diseases including CF (van der Vliet et al., 1997). However, we were unable to detect any changes in psoriasin protein expression in either cell line over the concentrations of t-BOOH we were able to investigate. Our findings contrast those of Carlsson et al. (2005), who reported an increase in psoriasin after a short term exposure to H2 O2 and proposed the protein may be cytoprotective against ROS-induced cell death in mammary epithelium. However, when we replicated the oxidant stress protocol used by Carlsson et al. (2005), we were unable to detect any differences in our model systems. Instead, we decided to move to a more stable oxidant stressor, t-BOOH, which should persist in an active form for longer, enabling us to investigate chronic oxidant stress exposure. Interestingly, the CF cells succumbed to oxidant stress at a much lower concentration of tBOOH than the non-CF, suggesting that their absence of a functional

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CFTR channel may be detrimental to their overall well-being. However, such significant cell death was observed above 500 ␮M and 100 ␮M t-BOOH in the 16HBEo- and CFBE41o- cells, respectively that it was impossible to extract protein for western blotting. It is possible, of course, that psoriasin expression would be altered at these high concentrations, but the physiological significance is limited. Our finding of a lack of effect on psoriasin expression after 24 h exposure contradicts our initial hypothesis and makes it extremely unlikely, in our opinion, that psoriasin is playing an adaptive role to combat deleterious oxidant stress, at least in 16HBE14o- cells. Finally, since psoriasin has reported anti-microbial activity, we wished to investigate whether increasing its expression would affect how these cell lines treated a bacterial strain of relevance to CF lung disease. The CF lung is often chronically infected due to colonization by bio-film forming, mucoid, strains of P. aeruginosa (Høiby, 2011); therefore we chose to investigate a mucoid strain of P. aeruginosa isolated from a CF patient (strain 8821). Airway epithelial cells are the first line of defence against inhaled bacteria and because we were interested in the innate ability of these cells to cope with an infection burden, we additionally chose to investigate only their response to infection in a short time-frame (i.e. 90 min). Initially, when P. aeruginosa were applied to both cell lines at an equivalent amount (MOI of 25), the numbers of bacteria internalized by the CFBE41o- line were significantly higher than the 16HBE14o- cells. Furthermore, transfection of a human psoriasin construct into both cell lines, resulting in increased levels of psoriasin protein, resulted in significantly decreased P. aeruginosa internalization when compared to controls. Therefore, we conclude that the more psoriasin protein expressed by a cell, the less P. aeruginosa will be internalized. Previous studies have reported that various strains of P. aeruginosa can enter airway epithelial cells; however, the mechanism of entry and the biological consequences of internalized bacteria are unclear. Plotkowski et al. (1999) determined that P. aeruginosa internalization into airway epithelial cells depended not upon the presence of functional CFTR at the cell membrane, but rather whether the cells were fully polarized and in possession of effective tight junction complexes. Although normal airway epithelial cells are normally highly resistant to apoptosis, infection with P. aeruginosa can lead to apoptosis in susceptible cell lines, such as those that do not develop tight junctions (Rajan et al., 2000). In general it would appear that poorly polarized cells and/or damaged epithelial are more susceptible to P. aeruginosa infection. It is important to note that cells were not polarized in the present study, thus eliminating the potential effects of tight junction complexes Cannon et al. (2003) reported there is an inherent defect in the ability of CF cells to undergo apoptosis following P. aeruginosa infection. Their proposed hypothesis was that rapid apoptosis of infected airway epithelial cells was an essential part of host defence, permitting infected cells (and thus internalized bacteria) to be cleared from the lungs. CF cells displayed a delayed apoptotic response to P. aeruginosa infection, which permitted the bacteria to evade host defence mechanisms and remain in the infected airway cells for longer. Darling et al. (2004) added to this work by reporting that P. aeruginosa are internalized into CFBE41o- cells, which possess F508- CFTR, where they can survive for extended periods without harming the infected cells, again escaping innate host defence mechanisms. These authors proposed that the presence of a mutant CFTR, such as F508- CFTR, leads to an increased susceptibility of infection by P. aeruginosa, which can remain in the cells for longer. Although the aims of the present study were not to investigate the effects of mutant/wild type CFTR on Pseudomonas internalization, the results we find do broadly fit into the hypotheses described above. Transfection of human psoriasin into either cell line resulted in a significant decrease in the number of colony forming, viable, bacteria we were able to recover when compared to controls. This

indicates an increased number of bacteria were internalized in the presence of enhanced psoriasin expression. Psoriasin is predominately a secreted protein, thus one interpretation of our data is that the higher the level of psoriasin within the cell, the more will be secreted, which prevents bacteria from becoming internalized. Conversely, the lower the levels of this protein, the more bacteria become internalized permitting them to remain within the cells for longer. A significant limitation of this approach is that it only measures bacterial internalization and we have not investigated any extracellular effects against bacteria. Furthermore, we have not investigated bacterial killing, which would be a true indication that this protein is an effective anti-microbial. The initial report of psoriasin’s anti-bacterial activity reported that it possessed a potent ability to kill several strains of E. coli, with more modest anti-microbial effects against S. aereus and P. aeruginosa (Glaser et al., 2005). Finally, we have only investigated one strain of P. aeruginosa in the present study, a strain not examined in the initial report. However, our data, while modest, do support the hypothesis that increased levels of psoriasin can result in protection against internalization of mucoid P. aeruginosa and that, while non-physiological, over-expressing this protein results in a change in the way airway epithelial cells cope with this pathogen. Psoriasin is only one of a large number of anti-microbial agents released from airway epithelial cells (Hiemstra, 2007; Hata and Gallo, 2008) and it is unclear how this protein would interact with other anti-microbial proteins in the airway surface liquid. However, we believe that our data suggests that increased investigation of this protein may provide another piece of information as to how CF cells are more susceptible to bacterial infection. In conclusion, we describe for the first time the expression of psoriasin in cell culture models of normal and CF airway epithelial cell lines, as well as its modulation by exposure to oxidant stress, IL-22 and other inflammatory cytokines. While the present work does not definitively ascribe a biological role for psoriasin in airway epithelia, the low levels in CFBE41o- cells hint at the possibility that the role of this protein may be compromised in the CF airway, and may be worthy of further investigation in native cells. Furthermore, we demonstrate that increasing psoriasin expression is effective in decreasing internalization of a mucoid P. aeruginosa strain, the first time this has been demonstrated using model human airway epithelial cells. Acknowledgments We wish to thank Christina Jones for excellent technical assistance, Dr Eileen Denovan-Wright for assistance with the qPCR studies, Dr Andrew Stadnyk for critical reading of the manuscript, and Drs Tong-Jun Lin and Dr Nikhil Thomas for assistance with the P. aeruginosa work. This work was supported entirely by Cystic Fibrosis Canada. References Aujla, S.J., Chan, Y.R., Zheng, M., Fei, M., Askew, D.J., Pociask, D.A., Reinhart, T.A., McAllister, F., Edeal, J., Gaus, K., Husain, S., Kreindler, J.L., Dubin, P.J., Pilewski, J.M., Myerburg, M.M., Mason, C.A., Iwakura, Y., Kolls, J.K., 2008. IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia. Nature Medicine 14, 275–281. Aujla, S.J., Kolls, J.K., 2009. IL-22: a critical mediator in mucosal host defense. Journal of Molecular Medicine 7, 451–454. Bonfield, T.L., Konstan, M.W., Berger, M., 1999. Altered respiratory epithelial cell cytokine production in cystic fibrosis. Journal of Allergy and Clinical Immunology 104, 72–78. Broome, A.M., Ryan, D., Eckert, R.L., 2003. S100 protein subcellular localization during epidermal differentiation and psoriasis. Journal of Histochemistry and Cytochemistry 51, 675–685. Cannon, C.L., Kowalski, M.P., Stopak, K.S., Pier, G.B., 2003. Pseudomonas aeruginosainduced apoptosis is defective in respiratory epithelial cells expressing mutant

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