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Prolactin stimulates the internalization of Staphylococcus aureus and modulates the expression of inflammatory response genes in bovine mammary epithelial cells
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Veterinary Immunology and Immunopathology 121 (2008) 113–122 www.elsevier.com/locate/vetimm
Prolactin stimulates the internalization of Staphylococcus aureus and modulates the expression of inflammatory response genes in bovine mammary epithelial cells Angelina Gutie´rrez-Barroso a,1, Jose´ L. Anaya-Lo´pez a,1, Leticia Lara-Za´rate a, Pedro D. Loeza-Lara b, Joel E. Lo´pez-Meza a, Alejandra Ochoa-Zarzosa a,* a
Centro Multidisciplinario de Estudios en Biotecnologı´a-Facultad de Medicina Veterinaria y Zootecnia, Universidad Michoacana de San Nicola´s de Hidalgo, Apdo. Postal 53, Administracio´n Chapultepec, C.P. 58262, Morelia, Michoaca´n, Me´xico b Facultad de Biologı´a., Universidad Michoacana de San Nicola´s de Hidalgo, Morelia, Michoaca´n, Me´xico Received 27 March 2007; received in revised form 11 July 2007; accepted 26 September 2007
Abstract The incidence of mastitis in dairy cattle is highest at the drying off period and parturition, which are characterized by high levels of the lactogenic hormone prolactin (PRL). One of the most frequently isolated contagious pathogens causing mastitis is Staphylococcus aureus. However, the role of PRL on S. aureus infection in mammary epithelium has not been studied. In this work we evaluated the effect of bovine PRL (bPRL) on S. aureus internalization in a primary culture of bovine mammary epithelial cells (bMEC) and on the expression of cytokine and innate immune response genes. Our data show that 5 ng/mL bPRL enhances 3-fold the internalization of S. aureus (ATCC 27543) into bMEC. By RT-PCR analysis, we showed that bPRL is able to upregulate the expression of tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b) and inducible nitric oxide synthase (iNOS) mRNAs. However, bPRL together with S. aureus did not modify the expression of TNF-a and iNOS mRNAs, while it downregulated the expression of b-defensin and IL-1b mRNAs, as well as nitric oxide production, suggesting that infection and bPRL together can inhibit elements of the host immune response. To our knowledge, this is the first report that shows a role of bPRL during the internalization of S. aureus into bMEC. # 2007 Elsevier B.V. All rights reserved. Keywords: Prolactin; Mastitis; Mammary epithelium; Staphylococcus aureus; Cytokines; iNOS
1. Introduction Bovine mastitis is the most important infectious disease of dairy cattle, affecting both the quality and quantity of milk produced in the world. This disease is * Corresponding author. Tel.: +52 443 295 8029; fax: +52 443 295 8029. E-mail addresses: [email protected], [email protected] (A. Ochoa-Zarzosa). 1 These authors contributed equally to this work. 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.09.007
characterized by an inflammatory response of the mammary tissue caused by bacterial and fungal infections. The highest incidence of udder infection and the establishment of mastitis occur during the drying off period and around calving (Burvenich et al., 1999; Burton and Erskine, 2003). These periods are characterized by important physiological changes related to milk production and metabolism. However, the functions of lactogenic hormones such as prolactin (PRL) during the udder infection and the establishment of mastitis remain unclear. Recently, it has been
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reported that during bovine experimentally induced chronic mastitis plasma PRL concentrations did not differ between healthy and infected cows (Hockett et al., 2000; Boutet et al., 2007). Through bovine lactation and calving, PRL stimulates metabolic functions of epithelial cells, maintains the concentrations of mRNA for synthesis of milk proteins and influences the milk production together with growth hormone (GH) (Neville et al., 2002; SvennerstenSjaunja and Olsson, 2005). In addition to the metabolic and lactogenic role of PRL, this hormone has been considered as a cytokine able to modulate the inflammatory response of the mammary epithelium (Brand et al., 2004; Boutet et al., 2007). The most common infective agents causing mastitis in dairy cattle are pathogenic coliform environmental bacteria and Staphylococcus species acquired by contagious transfer (Watts, 1988; Yancey, 1999). Staphylococcus aureus is one of the most frequently isolated contagious pathogen causing mastitis, which may result in chronic infections characterized by the colonization of mammary tissue and the survival of the bacteria within epithelial cells (Kerro-Dego et al., 2002). The invasive ability of bacteria can be evaluated in vitro by measuring their capacity to internalize in cultures of bovine mammary epithelial cells. Secretory epithelial cells respond to bacterial intrusion and play a major role in the initiation of inflammation (Burton and Erskine, 2003; Alluwaimi, 2004). The in vitro challenge of bovine mammary epithelial cells (bMEC) with lipoteichoic acid, a gram-positive bacterial cell wall component, induces an up-regulation of the gene expression for cytokines IL-1b, TNF-a and IL-8 and for the antimicrobial peptide b-defensin (Strandberg et al., 2005). Additionally, under inflammatory conditions, bMEC are able to produce nitric oxide (NO) (Boulanger et al., 2001), which is formed by the inducible nitric oxide synthase (iNOS) in mammary gland (Onona and Inano, 1998). In immortalized bovine mammary epithelial cells (MAC-T), PRL enhances the expression of several cytokines and modulates the expression of iNOS in fibroblasts (Corbacho et al., 2003; Boutet et al., 2007). However, there is no evidence that PRL regulates the internalization of S. aureus into mammary epithelium. The main purpose of this work was to evaluate the effect of bovine PRL (bPRL) on the internalization of S. aureus into a primary culture of bMEC. Additionally, we analyzed the effect of bPRL on the expression of TNF-a, IL-1b, b-defensin, and iNOS mRNAs, as well as on NO production in bMEC infected with S. aureus.
2. Materials and methods 2.1. Culture of primary bovine mammary epithelial cells The isolation of bMEC was performed from alveolar tissue of udders of lactating cows as described previously (Anaya-Lo´pez et al., 2006). Cells from passages second to eighth were cultured in Petri dishes (Corning–Costar) in growth medium (GM) composed by Dulbecco’s modified Eagles’s medium/nutrient mixture F-12 Ham (DMEM/F-12, Sigma) supplemented with 10% fetal calf serum (FCS) (Equitech-Bio), 10 mg/mL insulin (Sigma), 5 mg/mL hydrocortisone (Sigma), 100 U/mL penicillin and streptomycin (100 mg/mL) and 1 mg/mL amphotericin B (Invitrogen). 2.2. Invasion assays The American Type Culture Collection (ATCC) S. aureus subsp. aureus 27543 (kindly donated by V.M. Baizabal-Aguirre, CMEB-FMVZ-UMSNH, Me´xico) strain isolated from a case of clinical mastitis was used in this study. Bovine prolactin (bPRL) was generously provided by C. Clapp (Instituto de Neurobiologı´a, UNAM, Me´xico). Polarized monolayers of bMEC were created on plates coated with 6–10 mg/ cm2 rat-tail type I collagen (Sigma). Prior to invasion assays bMEC were incubated with different concentrations of bPRL (1–50 ng/mL) dissolved in GM without antibiotics and serum for 24 h. Then, confluent bMEC monolayers in 24-well plates (Corning–Costar) containing 2 105 cells/well were infected with a multiplicity of infection (MOI) of 30:1 bacteria per cell. For this, bMEC monolayers were washed three times with phosphate buffer saline (PBS, pH 7.4) and inoculated with 500 mL of bacterial suspensions to a density of 6 106 CFU/mL in Luria Bertani (LB) broth from overnight cultures, and incubated for 2 h in 5% CO2 at 37 8C. After infection, bMEC monolayers were washed three times with PBS and incubated in GM without serum supplemented with 50 mg/mL gentamicin (invasion assay medium) for 2 h at 37 8C to eliminate extracellular bacteria. Subsequently, bMEC monolayers were washed three times with PBS, detached with trypsin–EDTA (Sigma) and lysed with 250 mL of sterile distilled water. bMEC lysates were diluted 100-fold, plated on LB agar for triplicate and incubated overnight at 37 8C. Gentamicin-killed bacteria were used as negative control and were prepared using 50 mg/mL gentamicin (Sigma) for 2 h at 37 8C previous to
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challenge. The number of CFU was determined by the standard colony counting technique. Simultaneously, the number of bMEC cultured in each well was calculated for each invasion assay using a haemocytometer. The data are presented as the ratio of CFU recovered per bMEC. For the microscopic analysis of associated bacteria and RT-PCR analysis, bMEC were treated only with 5 ng/mL bPRL for 24 h and invasion assays were performed as described with a MOI of 30:1 bacteria. For microscopic analysis, the bMEC were grown on glass coverslips previously coated with collagen. After infection, bMEC were washed three times with PBS and fixed in 4% formaldehyde–PBS for 10 min at room temperature and visualized by light microscopy (Leica Microsystems). The number of associated bacteria was estimated by counting 60 fields. 2.3. Production of bPRL antibodies and purification of immunoglobulin G (IgG) Polyclonal antibodies against bPRL were generated with the purpose to determine a specific effect for the hormone. Antibodies were produced purifying bPRL (a 23 kDa band) in 10% polyacrylamide gels visualized by Coomassie blue. Band was excised and immediately equilibrated with PBS pH 7.4. This SDS-PAGE purified protein was injected subcutaneously to female New Zealand white rabbits. The immunizations were performed by weekly intervals, until completion of a total of 30–40 mg of protein per rabbit. Blood was collected from the auricular vein 2 weeks after the last injection and the serum was separated. The level of antiserum sensitivity was determined by dot immunoblot. IgGs from antiserum were purified on a protein A-Sepharose column (Sigma). Immune serum was added to columns equilibrated and washed with PBS (pH 8). IgGs were eluted out with 0.1 M sodium acetate (pH 3), into tubes with 1 M Tris– HCl (pH 8) to neutralize acid. The concentration of IgGs was determined by the Bradford method. In order to inhibit the effect of the hormone, during invasion assays bPRL and IgGs were incubated together with shaking at room temperature 24 h prior to infection. 2.4. Determination of nitrite Nitric oxide (NO) secreted by bMEC to culture medium was evaluated by measuring the nitrite concentration (NO2) in cell-free conditioned media (invasion assay media) using the Greiss reaction. Briefly, the Greiss reagent was freshly prepared by mixing equal volumes of stock solution A (10% sulfanilamide, 40% phosphoric acid, Sigma) and stock
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solution B [1% N-(1-naphthyl) ethylenediamine dihydrochloride, Sigma] before the measurement of nitrite. One part of the reagent mixture was transferred into seven parts of the conditioned media and absorbance at 595 nm was determined in a spectrophotometer (BioRad). The nitrite concentration was estimated from a standard curve prepared with NaNO2 (Sigma) in PBS (pH 7.4). NO production was evaluated in the conditioned media from the bMEC that were harvested for RNA isolation and RT-PCR analysis. 2.5. RNA isolation and RT-PCR bMEC total RNA (5 mg) was extracted with Trizol (Invitrogen) according to manufacturer’s instructions, and then was used to synthesize cDNA from bMEC challenged with S. aureus, treated with bPRL or both. Genomic DNA contamination was removed from RNA sample by DNase I treatment (Sigma). Reverse transcription (RT) reaction was performed in 20 mL containing 25 mg/mL Oligo d(T) (Invitrogen) and 500 nM dNTPs (Invitrogen). The reaction was incubated at 65 8C for 5 min, and then immediately transferred to ice. Then, 1 First Strand Buffer (Invitrogen), 10 mM dithiothreitol and 2 U/mL RNAse inhibitor (Invitrogen) were added to the reaction mixture and incubated at 37 8C for 2 min. Finally, 10 U/mL M-MLV reverse transcriptase (Invitrogen) was added and the mixture was incubated again at 37 8C for 50 min, followed by 90 8C for 2 min. The primers (Invitrogen) used to amplify bovine TNF-a, IL-1b, b-defensin, iNOS and k-casein transcripts are shown in Table 1. Primers to amplify bactin (Table 1) were used to verify the integrity of the RNA as well as the efficiency of the RT. Each polymerase chain reaction (PCR) contained 2 mL cDNA, 200 nM dNTPs, 0.4 mmol each primer, 0.5 U Taq DNA Polymerase (Invitrogen) and 1 PCR Buffer (Invitrogen). The PCR products were visualized by electrophoresis in agarose gel (2%). Specificity of PCR products was determined by sequence analysis. 2.6. Densitometric analysis Volume analysis of PCR products was performed by using the RFLP Scan software (v. 2.1, Scanalytics) at a resolution setting of 300–600 dpi. 2.7. Data analysis Data from invasion assays, densitometric analysis and NO production were obtained from three independent experiments performed each in triplicate. The
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Table 1 PCR primers used in this study Specificity
Primer
Sequence
Fragment size (bp)
Annealing temperature (8C) and cycle number
Reference
Bovine TNF-a
Forward Reverse
50 -CTGGTTCAGACACTCAGGTCCT-30 50 -GAGGTAAAGCCCGTCAGCA-30
183
60/35
Strandberg et al. (2005)
Bovine IL-1b
Forward Reverse
50 -AAATGAACCGAGAAGTGGTGTT-30 50 -TTCCATATTCCTCTTGGGGTAGA-30
185
55/35
Strandberg et al. (2005)
Bovine b-defensin
Forward Reverse
50 -TCTTCTGGTCCTGTCTGCT-30 50 -CCGAACAGGTGCCAATCTGT-30
130
55/35 Strandberg et al. (2005)
0
0
Bovine iNOS
Forward Reverse
5 -GGAAGCAGTAACAAAGGAGATAG3 50 -CATAGCGGATGAGCTGGGCG-30
282
60/40
Boulanger et al. (2001)
Bovine k-casein
Forward Reverse
50 -AGCAAGAGCTGACGGTCACAA-30 50 -TGGCAGGCACAGTATTTGACA-30
350
58/40
Accession no. AY899918
b-actin
Forward Reverse
50 -ATGGTGGGCATGGGTCAGAA-30 50 -TCATACTCCTGCTTGCTGAT-30
900
55/25
Torner et al. (1999)
estimated values from densitometric analysis of PCR were expressed as relation to b-actin. Data were compared by analysis of variance and Student’s t-test. The results are reported as means the standard errors (S.E.). P values of <0.05 were considered significant. 3. Results 3.1. Effect of bPRL on the internalization of S. aureus by bMEC To investigate the effect of bPRL on the internalization process of S. aureus by mammary epithelial cells, bMEC cultures were treated with different concentrations of bPRL (1–50 ng/mL) during 24 h previous to invasion assays, and then were infected for 2 h. In agreement with the values of CFU obtained, the internalization of S. aureus by bMEC was stimulated 3-fold by 5 ng/mL of bPRL (Fig. 1a, P < 0.05), whereas no significant effect at lower or higher concentrations was observed. To confirm that this effect was specific for bPRL, bMEC were incubated with antibodies against bPRL as described in Section 2, obtaining internalized CFU similar to control bMEC (Fig. 1b, P < 0.05). These data were confirmed quantifying the percentage of bMEC with associated bacteria and the number of bacteria associated to bMEC in each condition by light microscopy evaluation. The treatment of bMEC with bPRL (5 ng/mL) has no effect over the number of cells with associated bacteria (Fig. 2a). However, the number of S. aureus associated
to bMEC was stimulated by bPRL (5 ng/mL). Approximately 75% of the bMEC contained more than 20 bacteria associated per cell (Fig. 2b and d). In contrast, only 45% of control cells showed a similar number of associated bacteria (Fig. 2b and c, P < 0.05). When bMEC were challenged with killed S. aureus, the number of bacteria associated to bMEC was not modified by bPRL (Fig. 2e). 3.2. Analysis of mRNA expression of proinflammatory cytokines The expression of cytokines by bovine mammary epithelium has been reported elsewhere (Alluwaimi, 2004; Wellnitz and Kerr, 2004; Strandberg et al., 2005). In addition, it has been shown that PRL induces the expression of these cytokines in an immortalized bovine mammary epithelial cell line (MAC-T cells) (Boutet et al., 2007). However, the effect of bPRL and infection on cytokines expression has not been explored. We determined the expression of TNF-a and IL-1b mRNAs by RT-PCR in non-infected and infected bMEC with S. aureus during 2 h (Fig. 3). bPRL (5 ng/mL) significantly up-regulated the expression of TNF-a mRNA, whereas did not modify IL-1b mRNA expression in non-infected bMEC (Fig. 3b and d). Interestingly, S. aureus infection induced the expression of TNF-a (6fold, P < 0.05), which was not modified in the presence of bPRL. In addition, bPRL together with S. aureus infection down-regulated significantly IL-1b mRNA expression (4-fold, P < 0.05).
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Fig. 1. bPRL stimulates the internalization of Staphylococcus aureus in bMEC. (a) Effect of different concentrations of bPRL on the internalization of S. aureus by bMEC. The number of internalized bacteria is represented by the ratio of CFU/bMEC recovered after lysis of bMEC. (b) The effect of 5 ng/mL bPRL was reversed with 10 mg IgGs anti-bPRL. Each bar shows the mean of triplicates S.E. of three independent experiments. The symbols ‘‘*’’ and ‘‘**’’ indicate significant changes (P < 0.05) in relation to control and 5 ng/mL bPRL, respectively.
3.3. Analysis of mRNA expression of innate immune response genes The expression of b-defensin and iNOS has been reported in bovine mammary epithelial cells (Onona and Inano, 1998; Boulanger et al., 2001; Strandberg et al., 2005), but their regulation by bPRL remains unknown. We analyzed the expression of b-defensin and iNOS mRNAs by RT-PCR in non-infected and infected bMEC with S. aureus during 2 h (Fig. 4). bMEC treated with bPRL (5 ng/mL) under noninfection conditions showed a down-regulated expression of b-defensin mRNA (2-fold, Fig. 4b, P < 0.05), whereas the expression of iNOS mRNA showed an increase of 2-fold (Fig. 4d, P < 0.05). Likewise, bMEC under infection conditions showed an increase in the expression of b-defensin and iNOS mRNA (2-fold, Fig. 4b, P < 0.05) in relation to control non-infected. Also, bMEC under infection conditions and bPRL (5 ng/mL) showed a significant
reduction in b-defensin mRNA expression (40-fold, P < 0.05), whereas the iNOS mRNA expression was not modified. To determine if the infection modifies the expression of milk protein genes, the mRNA expression of k-casein was analyzed. k-casein expression was up-regulated in bMEC treated with bPRL (6-fold, P < 0.05) (Fig. 4f). In addition, kcasein was induced upon infection (3-fold), whereas infection and bPRL did not change its expression in relation to bPRL alone. 3.4. NO production by bMEC The production of NO by bovine mammary epithelial cells and whole mammary glands has been reported during inflammatory conditions (Boulanger et al., 2001; Komine et al., 2004). Besides, the regulation of NO by PRL has been demonstrated in mouse mammary epithelial cells (Bolander, 2001, 2002). However, the production of NO by bMEC in
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Fig. 2. Effect of bPRL on the number of S. aureus associated to bMEC. (a) Percentage of bMEC with associated bacteria without (control) or with 5 ng/mL bPRL. (b) Percentage of bMEC with different number of S. aureus associated without (control) or with 5 ng/mL bPRL. Each bar shows the mean of 60 fields counted S.E. of three independent experiments. The symbol ‘‘*’’ indicates significant changes (P < 0.05) in relation to the same number of bacteria from control. Light microscopic photographs that show the association of S. aureus to control bMEC (c) or treated with 5 ng/mL bPRL (d). Arrows indicate bacteria. Scale bar: 1 mm. S. aureus killed with gentamicin was not able to associate to control bMEC nor treated with 5 ng/mL bPRL, whereas the number of live-associated bacteria increased in the presence of the hormone. In each condition 30 fields were counted (e). The symbol ‘‘*’’ indicates significant changes (P < 0.05) in relation to the same number of bacteria from control.
response to bPRL and infection has not been explored. In Table 2 we showed the amount of NO produced by bMEC during infection conditions, measured as nitrite concentration (NO2) in cell-free conditioned media. bMEC treated with bPRL (5 ng/mL) under noninfection conditions showed an increase in NO production (3-fold). Similarly, bMEC under S. aureus infection released significant amounts of NO in relation to non-infected cells (12-fold, P < 0.05). Interestingly, when bMEC were infected with S. aureus and treated with bPRL, the levels of NO released were significantly reduced in relation to infection alone (P < 0.05).
4. Discussion The fact that the incidence of clinical and subclinical mastitis is highest at drying off period and around calving (Vangroenweghe et al., 2005), the role of the lactogenic and immunomodulatory hormone prolactin during the establishment of the disease and host response, remains unclear. In this study, we evaluated the role of bPRL during the in vitro infection of bovine mammary epithelial cells with S. aureus, which is one of the most frequently isolated pathogens causing clinical or subclinical mastitis (Kerro-Dego et al., 2002). Primary cultures of bovine mammary epithelium
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Fig. 3. Analysis of mRNA expression of cytokines genes. RT-PCR analysis that shows the expression of mRNAs for TNF-a (a) and IL-1b (c). The bMEC non-infected (control) were treated with 5 ng/mL bPRL (bPRL). Then, the bMEC were infected with S. aureus during 2 h either without bPRL (control infected) or with 5 ng/mL bPRL (bPRL infected). The respective RT-PCR analysis for b-actin is also shown in all conditions. The densitometric analyses for TNF-a and IL-1b expression are shown in (b) and (d), respectively. Each bar shows the mean of triplicates S.E. of three independent experiments. bPRL: bMEC treated with bovine prolactin (5 ng/mL). The symbol ‘‘*’’ indicates significant changes (P < 0.05) in relation to control non-infected bMEC. The symbol ‘‘**’’ indicates significant changes (P < 0.05) in relation to the same condition but non-infected bMEC (control or with bPRL).
have been used to model epithelial cell response to infections caused by S. aureus and to evaluate invasion ability of S. aureus strains (Wellnitz and Kerr, 2004; Hensen et al., 2000). In this work, the results obtained with the invasion assays indicate that bPRL can enhance the internalization of S. aureus into bMEC. This effect was detected at 5 ng/mL bPRL (Fig. 1a), which is in agreement with the physiological levels of bPRL reported in cattle during lactation (Hockett et al., 2000; Boutet et al., 2007). The stimulation of S. aureus internalization into bMEC is a specific effect for bPRL, because it was reversed with antibodies anti-bPRL (Fig. 1b). The bell-shaped hormone response curve observed (Fig. 1a), suggests that this effect can be mediated by the action of bPRL receptors located on bMEC, and corresponds to the binding mechanism proposed for several lactogenic hormones (Fuh et al., 1993). The integrin receptor a5b1 has been considered as the main host cell receptor that regulates the adhesion and invasion of S. aureus (Sinha et al., 1999). We blocked this integrin with latex beads covered with fibronectin prior to infection assays. Data revealed that S. aureus endocytosis into bMEC was not totally
abolished suggesting that integrin a5b1 is necessary for the endocytosis induced by bPRL but it is not the only mechanism (data not shown). On the other hand, adhesion and invasion of killed S. aureus has been demonstrated for several cell types, including endothelial and corneal epithelial cells (Sinha et al., 1999; Jett and Gilmore, 2002). However, in the present study we did not detect any changes in the number of killed S. aureus associated to bMEC (Fig. 2e), suggesting that other factors are necessary for endocytosis into bMEC in addition to the membrane adhesins that interact with host cell receptors (Sinha and Herrmann, 2005). PRL has been considered as a cytokine able to modulate the inflammatory response of the mammary epithelium (Brand et al., 2004; Boutet et al., 2007). Recently, Boutet et al. (2007) have demonstrated cytokine mRNA expression in MAC-T cells stimulated for 30 and 180 min with bPRL. Besides, Strandberg et al. (2005) have reported that in vitro challenge of bMEC with lipoteichoic acid induces an up-regulation of the gene expression of TNF-a, IL-1b, and IL-8. In order to analyze the expression of inflammatory response genes in bMEC under treatment with bPRL and infection conditions, we carried out RT-PCR
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Fig. 4. Analysis of mRNA expression of innate immune response genes. RT-PCR analysis that shows the expression of mRNAs for b-defensin (a), iNOS (c) and k-casein (e). The bMEC non-infected (control) were treated with 5 ng/mL bPRL (bPRL). Then, the bMEC were infected with S. aureus during 2 h either without bPRL (control infected) or with 5 ng/mL bPRL (bPRL infected). The respective RT-PCR analysis for b-actin is also shown in all conditions. The densitometric analyses for b-defensin, iNOS and k-casein expression are shown in (b), (d) and (f), respectively. Each bar shows the mean of triplicates S.E. of three independent experiments. bPRL: bMEC treated with bovine prolactin (5 ng/mL). The symbol ‘‘*’’ indicates significant changes (P < 0.05) in relation to control non-infected bMEC. The symbol ‘‘**’’ indicates significant changes (P < 0.05) in relation to the same condition but non-infected bMEC (control or with bPRL).
analysis of cytokines TNF-a and IL-1b. In agreement with Strandberg et al. (2005) and Boutet et al. (2007), bPRL (5 ng/mL) promoted a slightly up-regulation of TNF-a in bMEC non-infected, whereas S. aureus infection significantly enhanced the expression of TNF-a. Interestingly, the infection and bPRL together
reduced IL-1b mRNA and did not change TNF-a (Fig. 3). To our knowledge, the present findings represent the first evidence that bPRL and S. aureus infection down-regulate the expression for IL-1b in bMEC. In vitro studies using endothelial cells and the immortal cell line MAC-T indicate that the infection
Table 2 NO production by bMEC
Control bMEC infected with Staphylococcus aureus a b c
Without bPRL (mM S.E.M.)a
With 5 ng/mL bPRL (mM S.E.M.)
0.4111 0.0001 4.9526 0.792c
1.3799 0.31b 4.0443 0.0004b
Values are means S.E.M. of three independent experiments of NO secreted by bMEC, measured as nitrite concentration. Significantly different (P < 0.05) from the respective value without bPRL, as calculated by Student’s t-test. Significantly different (P < 0.05) from value for non-infected cells, as calculated by Student’s t-test.
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with S. aureus induced apoptosis (Menzies and Kourteva, 1998). Preliminary results of our laboratory demonstrated that bMEC start dying after 8 h of infection with S. aureus, but further experiments are necessary to determine if apoptosis is induced in our model. Under inflammatory conditions, bMEC are able to produce NO in mammary gland (Onona and Inano, 1998; Boulanger et al., 2001). Additionally, PRL modulates the expression of iNOS in fibroblasts (Corbacho et al., 2003). In agreement with these results, we detected an induction of iNOS mRNA in bMEC treated with bPRL and under infection conditions (Fig. 4d). In general, these results have correlation with NO production in bMEC. Thus, this is the first report that shows an induction for iNOS expression upon S. aureus infection in bMEC. Likewise, in addition to the effect of bPRL on iNOS mRNA expression, this hormone can be regulating other mechanisms necessary for NO production, for example Ca2+-dependent iNOS activation, as has been reported in mouse mammary epithelium (Bolander, 2002, 2005), which could explain the down-regulation in NO production in bMEC infected and treated with bPRL. The expression of b-defensin upon inflammatory conditions has been reported in bovine mammary epithelial cells (Strandberg et al., 2005), but its expression regulated by lactogenic hormones remains unknown. bMEC only treated with bPRL (5 ng/mL) and under infection conditions together with bPRL showed a down-regulated expression of b-defensin mRNA (Fig. 4b). On the other hand, bMEC only infected showed an increase in the expression of b-defensin. These results suggest that bPRL and infection regulate the expression of elements of host innate immune response such as defensins, but the triggered mechanisms remain unknown. Additionally, the k-casein expression was up-regulated by bPRL as well as by infection with S. aureus, suggesting that k-casein mRNA is induced through internalization (Fig. 4e). The expression of caseins in response to the activation of JAK/Stat pathway induced by PRL is well known (Groner, 2002). Additionally, the up-regulation of the JAK/Stat pathway by intracellular bacteria has been reported for the infection with Chlamydia trachomatis (Lad et al., 2005). Accordingly, it has been suggested that during lactation the low activity of NFkB might be important for the high level of expression of casein genes, which are mainly induced by the activation of JAK/Stat pathway (Geymayer and Doppler, 2000; Neville et al., 2002; Sheehy et al., 2004). NFkB activation induced by bPRL has been reported in MAC-
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T cell stimulated during 30 min with bPRL. In this work we treated bMEC with bPRL during 24 h prior to infection, and the hormone was present throughout in order to mimic in vivo conditions. Further research is necessary to determine if NFkB and JAK2/Stat5 signal transduction pathway are activated or repressed under the experimental conditions showed in this work. In conclusion, bPRL stimulates the internalization of viable S. aureus into bovine mammary epithelial cells. These cells were able to raise different innate immune responses to infection in the presence of the hormone, suggesting that bPRL together with S. aureus regulate diverse host innate immune elements. These results may help to explain the reduced innate immune response detected in cows with chronic mastitis attributable mainly to S. aureus (Lee et al., 2006). However, the role of bPRL in cows with coliform mastitis needs further investigation because during in vivo infections, cows challenged with Escherichia coli and S. aureus show different innate immune responses (Bannerman et al., 2004). Acknowledgments We thank Luz Torner-Aguilar for careful reading and helpful suggestions of the manuscript. J.L.A.L. and P.D.L.L. were supported by scholarships from CONACyT. This work was supported by grants from Coordinacio´n de la Investigacio´n Cientı´fica (UMSNH, CIC 14.1) and CONACyT (46400) to A.O.Z. and UMSNH, CIC 14.5 to J.E.L.M. References Alluwaimi, A.M., 2004. The cytokines of bovine mammary gland: prospects for diagnosis and therapy. Res. Vet. Sci. 77, 211–222. Anaya-Lo´pez, J.L., Contreras-Guzma´n, O.E., Ca´rabez-Trejo, A., Baizabal-Aguirre, V.M., Lo´pez-Meza, J.E., Valdez-Alarco´n, J.J., Ochoa-Zarzosa, A., 2006. Invasive potential of bacterial isolates associated with subclinical bovine mastitis. Res. Vet. Sci. 81, 358–361. Bannerman, D.D., Paape, M.J., Lee, J.W., Zhao, X., Hope, J.C., Rainard, P., 2004. Escherichia coli and Staphylococcus aureus elicit differential innate immune responses following intramammary infection. Clin. Diagn. Lab. Immunol. 11, 463–472. Bolander, F.F., 2001. The role of nitric oxide in the biological activity of prolactin in the mouse mammary gland. Mol. Cell. Endocrinol. 174, 91–98. Bolander, F.F., 2002. Prolactin activation of mammary nitric oxide synthase: molecular mechanisms. J. Mol. Endocrinol. 28, 45–51. Bolander, F.F., 2005. The compartmentalization of prolactin signaling in the mouse mammary gland. Mol. Cell. Endocrinol. 245, 105–110.
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Boulanger, V., Bouchard, L., Zhao, X., Lacasse, P., 2001. Induction of nitric oxide production by bovine mammary epithelial cells and blood leukocytes. J. Dairy Sci. 84, 1430–1437. Boutet, P., Sulon, J., Closset, R., Detilleux, J., Beckers, J.F., Bureau, F., Lekeux, P., 2007. Prolactin-induced activation of nuclear factor kB in bovine mammary epithelial cells: role in chronic mastitis. J. Dairy Sci. 90, 155–164. Brand, J.M., Frohn, C., Cziupka, K., Brockmann, C., Kirchner, H., Luhm, J., 2004. Prolactin triggers pro-inflammatory immune responses in peripheral immune cells. Eur. Cytokine Netw. 15, 99–104. Burton, J.L., Erskine, R.J., 2003. Immunity and mastitis: some new ideas for an old disease. Vet. Clin. Food Anim. 19, 1–45. Burvenich, C., Paape, M.J., Hoeben, D., Dosogne, H., Massart-Lee¨n, A.M., Blum, J.J., 1999. Modulation of the inflammatory reaction and neutrophil defense of the bovine lactating mammary gland by growth hormone. Domest. Anim. Endocrinol. 17, 149–159. Corbacho, A.M., Macotela, Y., Nava, G., Eiserich, J.P., Cross, C.E., Martı´nez de la Escalera, G., Clapp, C., 2003. Cytokine induction of prolactin receptors mediates prolactin inhibition of nitric oxide synthesis in pulmonary fibroblasts. FEBS Lett. 544, 171–175. Fuh, G., Colosi, P., Woods, W.I., Wells, J.A., 1993. Mechanismbase design of prolactin receptor antagonists. J. Biol. Chem. 268, 5376–5381. Geymayer, S., Doppler, W., 2000. Activation of NF-kB p50/p65 is regulated in the developing mammary gland and inhibits STAT5mediated b-casein gene expression. FASEB J. 14, 1159–1170. Groner, B., 2002. Transcription factor regulation in mammary epithelial cells. Domest. Anim. Endocrinol. 23, 25–32. Hensen, S.M., Pavicˇic´, M.J.A.P., Lohuis, J.A.C.M., Poutrel, B., 2000. Use of a bovine primary mammary epithelial cells for the comparison of adherence and invasion ability of Staphylococcus aureus strains. J. Dairy Sci. 83, 418–429. Hockett, M.E., Hopkins, F.M., Lewis, M.J., Saxton, A.M., Dowlen, H.H., Oliver, S.P., Schirck, F.N., 2000. Endocrine profiles of dairy cows following experimentally induced clinical mastitis during early lactation. Anim. Reprod. Sci. 58, 241–251. Jett, B.D., Gilmore, M.S., 2002. Internalization of Staphylococcus aureus by human corneal epithelial cells: role of bacterial fibronectin binding protein and host cell factors. Infect. Immun. 70, 4697–4700. Kerro-Dego, O., van Dijk, J.E., Nederbragt, H., 2002. Factors involved in the early pathogenesis of bovine Staphylococcus aureus mastitis with emphasis on bacterial adhesion and invasion. Rev. Vet. Q. 24, 181–198. Komine, K., Kuroishi, T., Komine, Y., Watanabe, K., Kobayashi, J., Yamaguchi, T., Kamata, S., Kumagai, K., 2004. Induction of nitric oxide production mediated tumor necrosis factor alpha on Staphylococcal enterotoxin C-stimulated bovine mammary gland cells. Clin. Diagn. Lab. Immun. 11, 203–210.
Lad, S.P., Fukuda, E.Y., Li, J., de la Maza, L.M., Li, E., 2005. Upregulation of the Jak/Stat1 signal pathway during Clamydia trachomatis infection. J. Immunol. 174, 7186–7193. Lee, J.W., Bannerman, D.D., Paape, M.J., Huang, M.K., Zhao, X., 2006. Characterization of cytokine expression in milk somatic cells during intramammary infections with Escherichia coli or Staphylococcus aureus by real time-PCR. Vet. Res. 37, 219–229. Menzies, B.E., Kourteva, I., 1998. Internalization of Staphylococcus aureus by endothelial cells induces apoptosis. Infect. Immun. 66, 5994–5998. Neville, M.C., McFadden, T.B., Forsyth, I., 2002. Hormonal regulation of mammary differentiation and milk secretion. J. Mammary Gland Biol. Neoplasia 7, 49–66. Onona, M., Inano, H., 1998. Localization of nitric oxide synthases and nitric oxide production in the rat mammary gland. J. Histochem. Cytochem. 46, 1269–1278. Sheehy, P.A., Della-Vedova, J.J., Nichols, K.R., Wynn, P.C., 2004. Hormone-dependent milk protein gene expression in bovine mammary gland explants from biopsies at different stages of pregnancy. J. Dairy Res. 71, 135–140. Sinha, B., Herrmann, M., 2005. Mechanism and consequences of invasion of endothelial cells by Staphylococcus aureus. Thromb. Haemost. 94, 266–277. Sinha, B., Franc¸ois, P.P., Nu¨ße, O., Foti, M., Hartford, O.M., Vaudaux, P., Foster, T.J., Lew, D.P., Herrmann, M., Krause, K.H., 1999. Fibronectin binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin a5b1. Cell. Microbiol. 1, 101–117. Strandberg, Y., Gray, C., Vuocolo, T., Donaldson, L., Broadway, M., Tellam, R., 2005. Lypopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells. Cytokine 31, 72–86. Svennersten-Sjaunja, K., Olsson, K., 2005. Endocrinology of milk production. Domest. Anim. Endocrinol. 29, 241–258. Torner, L., Nava, G., Duen˜as, Z., Corbacho, A., Mejı´a, S., Lo´pez, F., Cajero, M., Martı´nez de la Escalera, G., Clapp, C., 1999. Changes in the expression of neurohypophyseal prolactins during the estrous cycle and after estrogen treatment. J. Endocrinol. 161, 423–432. Vangroenweghe, F., Lamote, I., Burvenich, C., 2005. Physiology of the periparturient period and its relation to severity of clinical mastitis. Domest. Anim. Endocrinol. 29, 283–293. Watts, J.L., 1988. Etiological agents of bovine mastitis. Vet. Microbiol. 16, 41–66. Wellnitz, O., Kerr, D.E., 2004. Cryopreserved bovine mammary cells to model epithelial response to infection. Vet. Immunol. Immunopathol. 101, 191–202. Yancey, R.J., 1999. Vaccines and diagnostic methods for bovine mastitis: fact and fiction. Adv. Vet. Med. 41, 257–273.
Veterinary Immunology and Immunopathology 121 (2008) 113–122 www.elsevier.com/locate/vetimm
Prolactin stimulates the internalization of Staphylococcus aureus and modulates the expression of inflammatory response genes in bovine mammary epithelial cells Angelina Gutie´rrez-Barroso a,1, Jose´ L. Anaya-Lo´pez a,1, Leticia Lara-Za´rate a, Pedro D. Loeza-Lara b, Joel E. Lo´pez-Meza a, Alejandra Ochoa-Zarzosa a,* a
Centro Multidisciplinario de Estudios en Biotecnologı´a-Facultad de Medicina Veterinaria y Zootecnia, Universidad Michoacana de San Nicola´s de Hidalgo, Apdo. Postal 53, Administracio´n Chapultepec, C.P. 58262, Morelia, Michoaca´n, Me´xico b Facultad de Biologı´a., Universidad Michoacana de San Nicola´s de Hidalgo, Morelia, Michoaca´n, Me´xico Received 27 March 2007; received in revised form 11 July 2007; accepted 26 September 2007
Abstract The incidence of mastitis in dairy cattle is highest at the drying off period and parturition, which are characterized by high levels of the lactogenic hormone prolactin (PRL). One of the most frequently isolated contagious pathogens causing mastitis is Staphylococcus aureus. However, the role of PRL on S. aureus infection in mammary epithelium has not been studied. In this work we evaluated the effect of bovine PRL (bPRL) on S. aureus internalization in a primary culture of bovine mammary epithelial cells (bMEC) and on the expression of cytokine and innate immune response genes. Our data show that 5 ng/mL bPRL enhances 3-fold the internalization of S. aureus (ATCC 27543) into bMEC. By RT-PCR analysis, we showed that bPRL is able to upregulate the expression of tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b) and inducible nitric oxide synthase (iNOS) mRNAs. However, bPRL together with S. aureus did not modify the expression of TNF-a and iNOS mRNAs, while it downregulated the expression of b-defensin and IL-1b mRNAs, as well as nitric oxide production, suggesting that infection and bPRL together can inhibit elements of the host immune response. To our knowledge, this is the first report that shows a role of bPRL during the internalization of S. aureus into bMEC. # 2007 Elsevier B.V. All rights reserved. Keywords: Prolactin; Mastitis; Mammary epithelium; Staphylococcus aureus; Cytokines; iNOS
1. Introduction Bovine mastitis is the most important infectious disease of dairy cattle, affecting both the quality and quantity of milk produced in the world. This disease is * Corresponding author. Tel.: +52 443 295 8029; fax: +52 443 295 8029. E-mail addresses: [email protected], [email protected] (A. Ochoa-Zarzosa). 1 These authors contributed equally to this work. 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.09.007
characterized by an inflammatory response of the mammary tissue caused by bacterial and fungal infections. The highest incidence of udder infection and the establishment of mastitis occur during the drying off period and around calving (Burvenich et al., 1999; Burton and Erskine, 2003). These periods are characterized by important physiological changes related to milk production and metabolism. However, the functions of lactogenic hormones such as prolactin (PRL) during the udder infection and the establishment of mastitis remain unclear. Recently, it has been
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reported that during bovine experimentally induced chronic mastitis plasma PRL concentrations did not differ between healthy and infected cows (Hockett et al., 2000; Boutet et al., 2007). Through bovine lactation and calving, PRL stimulates metabolic functions of epithelial cells, maintains the concentrations of mRNA for synthesis of milk proteins and influences the milk production together with growth hormone (GH) (Neville et al., 2002; SvennerstenSjaunja and Olsson, 2005). In addition to the metabolic and lactogenic role of PRL, this hormone has been considered as a cytokine able to modulate the inflammatory response of the mammary epithelium (Brand et al., 2004; Boutet et al., 2007). The most common infective agents causing mastitis in dairy cattle are pathogenic coliform environmental bacteria and Staphylococcus species acquired by contagious transfer (Watts, 1988; Yancey, 1999). Staphylococcus aureus is one of the most frequently isolated contagious pathogen causing mastitis, which may result in chronic infections characterized by the colonization of mammary tissue and the survival of the bacteria within epithelial cells (Kerro-Dego et al., 2002). The invasive ability of bacteria can be evaluated in vitro by measuring their capacity to internalize in cultures of bovine mammary epithelial cells. Secretory epithelial cells respond to bacterial intrusion and play a major role in the initiation of inflammation (Burton and Erskine, 2003; Alluwaimi, 2004). The in vitro challenge of bovine mammary epithelial cells (bMEC) with lipoteichoic acid, a gram-positive bacterial cell wall component, induces an up-regulation of the gene expression for cytokines IL-1b, TNF-a and IL-8 and for the antimicrobial peptide b-defensin (Strandberg et al., 2005). Additionally, under inflammatory conditions, bMEC are able to produce nitric oxide (NO) (Boulanger et al., 2001), which is formed by the inducible nitric oxide synthase (iNOS) in mammary gland (Onona and Inano, 1998). In immortalized bovine mammary epithelial cells (MAC-T), PRL enhances the expression of several cytokines and modulates the expression of iNOS in fibroblasts (Corbacho et al., 2003; Boutet et al., 2007). However, there is no evidence that PRL regulates the internalization of S. aureus into mammary epithelium. The main purpose of this work was to evaluate the effect of bovine PRL (bPRL) on the internalization of S. aureus into a primary culture of bMEC. Additionally, we analyzed the effect of bPRL on the expression of TNF-a, IL-1b, b-defensin, and iNOS mRNAs, as well as on NO production in bMEC infected with S. aureus.
2. Materials and methods 2.1. Culture of primary bovine mammary epithelial cells The isolation of bMEC was performed from alveolar tissue of udders of lactating cows as described previously (Anaya-Lo´pez et al., 2006). Cells from passages second to eighth were cultured in Petri dishes (Corning–Costar) in growth medium (GM) composed by Dulbecco’s modified Eagles’s medium/nutrient mixture F-12 Ham (DMEM/F-12, Sigma) supplemented with 10% fetal calf serum (FCS) (Equitech-Bio), 10 mg/mL insulin (Sigma), 5 mg/mL hydrocortisone (Sigma), 100 U/mL penicillin and streptomycin (100 mg/mL) and 1 mg/mL amphotericin B (Invitrogen). 2.2. Invasion assays The American Type Culture Collection (ATCC) S. aureus subsp. aureus 27543 (kindly donated by V.M. Baizabal-Aguirre, CMEB-FMVZ-UMSNH, Me´xico) strain isolated from a case of clinical mastitis was used in this study. Bovine prolactin (bPRL) was generously provided by C. Clapp (Instituto de Neurobiologı´a, UNAM, Me´xico). Polarized monolayers of bMEC were created on plates coated with 6–10 mg/ cm2 rat-tail type I collagen (Sigma). Prior to invasion assays bMEC were incubated with different concentrations of bPRL (1–50 ng/mL) dissolved in GM without antibiotics and serum for 24 h. Then, confluent bMEC monolayers in 24-well plates (Corning–Costar) containing 2 105 cells/well were infected with a multiplicity of infection (MOI) of 30:1 bacteria per cell. For this, bMEC monolayers were washed three times with phosphate buffer saline (PBS, pH 7.4) and inoculated with 500 mL of bacterial suspensions to a density of 6 106 CFU/mL in Luria Bertani (LB) broth from overnight cultures, and incubated for 2 h in 5% CO2 at 37 8C. After infection, bMEC monolayers were washed three times with PBS and incubated in GM without serum supplemented with 50 mg/mL gentamicin (invasion assay medium) for 2 h at 37 8C to eliminate extracellular bacteria. Subsequently, bMEC monolayers were washed three times with PBS, detached with trypsin–EDTA (Sigma) and lysed with 250 mL of sterile distilled water. bMEC lysates were diluted 100-fold, plated on LB agar for triplicate and incubated overnight at 37 8C. Gentamicin-killed bacteria were used as negative control and were prepared using 50 mg/mL gentamicin (Sigma) for 2 h at 37 8C previous to
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challenge. The number of CFU was determined by the standard colony counting technique. Simultaneously, the number of bMEC cultured in each well was calculated for each invasion assay using a haemocytometer. The data are presented as the ratio of CFU recovered per bMEC. For the microscopic analysis of associated bacteria and RT-PCR analysis, bMEC were treated only with 5 ng/mL bPRL for 24 h and invasion assays were performed as described with a MOI of 30:1 bacteria. For microscopic analysis, the bMEC were grown on glass coverslips previously coated with collagen. After infection, bMEC were washed three times with PBS and fixed in 4% formaldehyde–PBS for 10 min at room temperature and visualized by light microscopy (Leica Microsystems). The number of associated bacteria was estimated by counting 60 fields. 2.3. Production of bPRL antibodies and purification of immunoglobulin G (IgG) Polyclonal antibodies against bPRL were generated with the purpose to determine a specific effect for the hormone. Antibodies were produced purifying bPRL (a 23 kDa band) in 10% polyacrylamide gels visualized by Coomassie blue. Band was excised and immediately equilibrated with PBS pH 7.4. This SDS-PAGE purified protein was injected subcutaneously to female New Zealand white rabbits. The immunizations were performed by weekly intervals, until completion of a total of 30–40 mg of protein per rabbit. Blood was collected from the auricular vein 2 weeks after the last injection and the serum was separated. The level of antiserum sensitivity was determined by dot immunoblot. IgGs from antiserum were purified on a protein A-Sepharose column (Sigma). Immune serum was added to columns equilibrated and washed with PBS (pH 8). IgGs were eluted out with 0.1 M sodium acetate (pH 3), into tubes with 1 M Tris– HCl (pH 8) to neutralize acid. The concentration of IgGs was determined by the Bradford method. In order to inhibit the effect of the hormone, during invasion assays bPRL and IgGs were incubated together with shaking at room temperature 24 h prior to infection. 2.4. Determination of nitrite Nitric oxide (NO) secreted by bMEC to culture medium was evaluated by measuring the nitrite concentration (NO2) in cell-free conditioned media (invasion assay media) using the Greiss reaction. Briefly, the Greiss reagent was freshly prepared by mixing equal volumes of stock solution A (10% sulfanilamide, 40% phosphoric acid, Sigma) and stock
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solution B [1% N-(1-naphthyl) ethylenediamine dihydrochloride, Sigma] before the measurement of nitrite. One part of the reagent mixture was transferred into seven parts of the conditioned media and absorbance at 595 nm was determined in a spectrophotometer (BioRad). The nitrite concentration was estimated from a standard curve prepared with NaNO2 (Sigma) in PBS (pH 7.4). NO production was evaluated in the conditioned media from the bMEC that were harvested for RNA isolation and RT-PCR analysis. 2.5. RNA isolation and RT-PCR bMEC total RNA (5 mg) was extracted with Trizol (Invitrogen) according to manufacturer’s instructions, and then was used to synthesize cDNA from bMEC challenged with S. aureus, treated with bPRL or both. Genomic DNA contamination was removed from RNA sample by DNase I treatment (Sigma). Reverse transcription (RT) reaction was performed in 20 mL containing 25 mg/mL Oligo d(T) (Invitrogen) and 500 nM dNTPs (Invitrogen). The reaction was incubated at 65 8C for 5 min, and then immediately transferred to ice. Then, 1 First Strand Buffer (Invitrogen), 10 mM dithiothreitol and 2 U/mL RNAse inhibitor (Invitrogen) were added to the reaction mixture and incubated at 37 8C for 2 min. Finally, 10 U/mL M-MLV reverse transcriptase (Invitrogen) was added and the mixture was incubated again at 37 8C for 50 min, followed by 90 8C for 2 min. The primers (Invitrogen) used to amplify bovine TNF-a, IL-1b, b-defensin, iNOS and k-casein transcripts are shown in Table 1. Primers to amplify bactin (Table 1) were used to verify the integrity of the RNA as well as the efficiency of the RT. Each polymerase chain reaction (PCR) contained 2 mL cDNA, 200 nM dNTPs, 0.4 mmol each primer, 0.5 U Taq DNA Polymerase (Invitrogen) and 1 PCR Buffer (Invitrogen). The PCR products were visualized by electrophoresis in agarose gel (2%). Specificity of PCR products was determined by sequence analysis. 2.6. Densitometric analysis Volume analysis of PCR products was performed by using the RFLP Scan software (v. 2.1, Scanalytics) at a resolution setting of 300–600 dpi. 2.7. Data analysis Data from invasion assays, densitometric analysis and NO production were obtained from three independent experiments performed each in triplicate. The
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Table 1 PCR primers used in this study Specificity
Primer
Sequence
Fragment size (bp)
Annealing temperature (8C) and cycle number
Reference
Bovine TNF-a
Forward Reverse
50 -CTGGTTCAGACACTCAGGTCCT-30 50 -GAGGTAAAGCCCGTCAGCA-30
183
60/35
Strandberg et al. (2005)
Bovine IL-1b
Forward Reverse
50 -AAATGAACCGAGAAGTGGTGTT-30 50 -TTCCATATTCCTCTTGGGGTAGA-30
185
55/35
Strandberg et al. (2005)
Bovine b-defensin
Forward Reverse
50 -TCTTCTGGTCCTGTCTGCT-30 50 -CCGAACAGGTGCCAATCTGT-30
130
55/35 Strandberg et al. (2005)
0
0
Bovine iNOS
Forward Reverse
5 -GGAAGCAGTAACAAAGGAGATAG3 50 -CATAGCGGATGAGCTGGGCG-30
282
60/40
Boulanger et al. (2001)
Bovine k-casein
Forward Reverse
50 -AGCAAGAGCTGACGGTCACAA-30 50 -TGGCAGGCACAGTATTTGACA-30
350
58/40
Accession no. AY899918
b-actin
Forward Reverse
50 -ATGGTGGGCATGGGTCAGAA-30 50 -TCATACTCCTGCTTGCTGAT-30
900
55/25
Torner et al. (1999)
estimated values from densitometric analysis of PCR were expressed as relation to b-actin. Data were compared by analysis of variance and Student’s t-test. The results are reported as means the standard errors (S.E.). P values of <0.05 were considered significant. 3. Results 3.1. Effect of bPRL on the internalization of S. aureus by bMEC To investigate the effect of bPRL on the internalization process of S. aureus by mammary epithelial cells, bMEC cultures were treated with different concentrations of bPRL (1–50 ng/mL) during 24 h previous to invasion assays, and then were infected for 2 h. In agreement with the values of CFU obtained, the internalization of S. aureus by bMEC was stimulated 3-fold by 5 ng/mL of bPRL (Fig. 1a, P < 0.05), whereas no significant effect at lower or higher concentrations was observed. To confirm that this effect was specific for bPRL, bMEC were incubated with antibodies against bPRL as described in Section 2, obtaining internalized CFU similar to control bMEC (Fig. 1b, P < 0.05). These data were confirmed quantifying the percentage of bMEC with associated bacteria and the number of bacteria associated to bMEC in each condition by light microscopy evaluation. The treatment of bMEC with bPRL (5 ng/mL) has no effect over the number of cells with associated bacteria (Fig. 2a). However, the number of S. aureus associated
to bMEC was stimulated by bPRL (5 ng/mL). Approximately 75% of the bMEC contained more than 20 bacteria associated per cell (Fig. 2b and d). In contrast, only 45% of control cells showed a similar number of associated bacteria (Fig. 2b and c, P < 0.05). When bMEC were challenged with killed S. aureus, the number of bacteria associated to bMEC was not modified by bPRL (Fig. 2e). 3.2. Analysis of mRNA expression of proinflammatory cytokines The expression of cytokines by bovine mammary epithelium has been reported elsewhere (Alluwaimi, 2004; Wellnitz and Kerr, 2004; Strandberg et al., 2005). In addition, it has been shown that PRL induces the expression of these cytokines in an immortalized bovine mammary epithelial cell line (MAC-T cells) (Boutet et al., 2007). However, the effect of bPRL and infection on cytokines expression has not been explored. We determined the expression of TNF-a and IL-1b mRNAs by RT-PCR in non-infected and infected bMEC with S. aureus during 2 h (Fig. 3). bPRL (5 ng/mL) significantly up-regulated the expression of TNF-a mRNA, whereas did not modify IL-1b mRNA expression in non-infected bMEC (Fig. 3b and d). Interestingly, S. aureus infection induced the expression of TNF-a (6fold, P < 0.05), which was not modified in the presence of bPRL. In addition, bPRL together with S. aureus infection down-regulated significantly IL-1b mRNA expression (4-fold, P < 0.05).
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Fig. 1. bPRL stimulates the internalization of Staphylococcus aureus in bMEC. (a) Effect of different concentrations of bPRL on the internalization of S. aureus by bMEC. The number of internalized bacteria is represented by the ratio of CFU/bMEC recovered after lysis of bMEC. (b) The effect of 5 ng/mL bPRL was reversed with 10 mg IgGs anti-bPRL. Each bar shows the mean of triplicates S.E. of three independent experiments. The symbols ‘‘*’’ and ‘‘**’’ indicate significant changes (P < 0.05) in relation to control and 5 ng/mL bPRL, respectively.
3.3. Analysis of mRNA expression of innate immune response genes The expression of b-defensin and iNOS has been reported in bovine mammary epithelial cells (Onona and Inano, 1998; Boulanger et al., 2001; Strandberg et al., 2005), but their regulation by bPRL remains unknown. We analyzed the expression of b-defensin and iNOS mRNAs by RT-PCR in non-infected and infected bMEC with S. aureus during 2 h (Fig. 4). bMEC treated with bPRL (5 ng/mL) under noninfection conditions showed a down-regulated expression of b-defensin mRNA (2-fold, Fig. 4b, P < 0.05), whereas the expression of iNOS mRNA showed an increase of 2-fold (Fig. 4d, P < 0.05). Likewise, bMEC under infection conditions showed an increase in the expression of b-defensin and iNOS mRNA (2-fold, Fig. 4b, P < 0.05) in relation to control non-infected. Also, bMEC under infection conditions and bPRL (5 ng/mL) showed a significant
reduction in b-defensin mRNA expression (40-fold, P < 0.05), whereas the iNOS mRNA expression was not modified. To determine if the infection modifies the expression of milk protein genes, the mRNA expression of k-casein was analyzed. k-casein expression was up-regulated in bMEC treated with bPRL (6-fold, P < 0.05) (Fig. 4f). In addition, kcasein was induced upon infection (3-fold), whereas infection and bPRL did not change its expression in relation to bPRL alone. 3.4. NO production by bMEC The production of NO by bovine mammary epithelial cells and whole mammary glands has been reported during inflammatory conditions (Boulanger et al., 2001; Komine et al., 2004). Besides, the regulation of NO by PRL has been demonstrated in mouse mammary epithelial cells (Bolander, 2001, 2002). However, the production of NO by bMEC in
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Fig. 2. Effect of bPRL on the number of S. aureus associated to bMEC. (a) Percentage of bMEC with associated bacteria without (control) or with 5 ng/mL bPRL. (b) Percentage of bMEC with different number of S. aureus associated without (control) or with 5 ng/mL bPRL. Each bar shows the mean of 60 fields counted S.E. of three independent experiments. The symbol ‘‘*’’ indicates significant changes (P < 0.05) in relation to the same number of bacteria from control. Light microscopic photographs that show the association of S. aureus to control bMEC (c) or treated with 5 ng/mL bPRL (d). Arrows indicate bacteria. Scale bar: 1 mm. S. aureus killed with gentamicin was not able to associate to control bMEC nor treated with 5 ng/mL bPRL, whereas the number of live-associated bacteria increased in the presence of the hormone. In each condition 30 fields were counted (e). The symbol ‘‘*’’ indicates significant changes (P < 0.05) in relation to the same number of bacteria from control.
response to bPRL and infection has not been explored. In Table 2 we showed the amount of NO produced by bMEC during infection conditions, measured as nitrite concentration (NO2) in cell-free conditioned media. bMEC treated with bPRL (5 ng/mL) under noninfection conditions showed an increase in NO production (3-fold). Similarly, bMEC under S. aureus infection released significant amounts of NO in relation to non-infected cells (12-fold, P < 0.05). Interestingly, when bMEC were infected with S. aureus and treated with bPRL, the levels of NO released were significantly reduced in relation to infection alone (P < 0.05).
4. Discussion The fact that the incidence of clinical and subclinical mastitis is highest at drying off period and around calving (Vangroenweghe et al., 2005), the role of the lactogenic and immunomodulatory hormone prolactin during the establishment of the disease and host response, remains unclear. In this study, we evaluated the role of bPRL during the in vitro infection of bovine mammary epithelial cells with S. aureus, which is one of the most frequently isolated pathogens causing clinical or subclinical mastitis (Kerro-Dego et al., 2002). Primary cultures of bovine mammary epithelium
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Fig. 3. Analysis of mRNA expression of cytokines genes. RT-PCR analysis that shows the expression of mRNAs for TNF-a (a) and IL-1b (c). The bMEC non-infected (control) were treated with 5 ng/mL bPRL (bPRL). Then, the bMEC were infected with S. aureus during 2 h either without bPRL (control infected) or with 5 ng/mL bPRL (bPRL infected). The respective RT-PCR analysis for b-actin is also shown in all conditions. The densitometric analyses for TNF-a and IL-1b expression are shown in (b) and (d), respectively. Each bar shows the mean of triplicates S.E. of three independent experiments. bPRL: bMEC treated with bovine prolactin (5 ng/mL). The symbol ‘‘*’’ indicates significant changes (P < 0.05) in relation to control non-infected bMEC. The symbol ‘‘**’’ indicates significant changes (P < 0.05) in relation to the same condition but non-infected bMEC (control or with bPRL).
have been used to model epithelial cell response to infections caused by S. aureus and to evaluate invasion ability of S. aureus strains (Wellnitz and Kerr, 2004; Hensen et al., 2000). In this work, the results obtained with the invasion assays indicate that bPRL can enhance the internalization of S. aureus into bMEC. This effect was detected at 5 ng/mL bPRL (Fig. 1a), which is in agreement with the physiological levels of bPRL reported in cattle during lactation (Hockett et al., 2000; Boutet et al., 2007). The stimulation of S. aureus internalization into bMEC is a specific effect for bPRL, because it was reversed with antibodies anti-bPRL (Fig. 1b). The bell-shaped hormone response curve observed (Fig. 1a), suggests that this effect can be mediated by the action of bPRL receptors located on bMEC, and corresponds to the binding mechanism proposed for several lactogenic hormones (Fuh et al., 1993). The integrin receptor a5b1 has been considered as the main host cell receptor that regulates the adhesion and invasion of S. aureus (Sinha et al., 1999). We blocked this integrin with latex beads covered with fibronectin prior to infection assays. Data revealed that S. aureus endocytosis into bMEC was not totally
abolished suggesting that integrin a5b1 is necessary for the endocytosis induced by bPRL but it is not the only mechanism (data not shown). On the other hand, adhesion and invasion of killed S. aureus has been demonstrated for several cell types, including endothelial and corneal epithelial cells (Sinha et al., 1999; Jett and Gilmore, 2002). However, in the present study we did not detect any changes in the number of killed S. aureus associated to bMEC (Fig. 2e), suggesting that other factors are necessary for endocytosis into bMEC in addition to the membrane adhesins that interact with host cell receptors (Sinha and Herrmann, 2005). PRL has been considered as a cytokine able to modulate the inflammatory response of the mammary epithelium (Brand et al., 2004; Boutet et al., 2007). Recently, Boutet et al. (2007) have demonstrated cytokine mRNA expression in MAC-T cells stimulated for 30 and 180 min with bPRL. Besides, Strandberg et al. (2005) have reported that in vitro challenge of bMEC with lipoteichoic acid induces an up-regulation of the gene expression of TNF-a, IL-1b, and IL-8. In order to analyze the expression of inflammatory response genes in bMEC under treatment with bPRL and infection conditions, we carried out RT-PCR
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Fig. 4. Analysis of mRNA expression of innate immune response genes. RT-PCR analysis that shows the expression of mRNAs for b-defensin (a), iNOS (c) and k-casein (e). The bMEC non-infected (control) were treated with 5 ng/mL bPRL (bPRL). Then, the bMEC were infected with S. aureus during 2 h either without bPRL (control infected) or with 5 ng/mL bPRL (bPRL infected). The respective RT-PCR analysis for b-actin is also shown in all conditions. The densitometric analyses for b-defensin, iNOS and k-casein expression are shown in (b), (d) and (f), respectively. Each bar shows the mean of triplicates S.E. of three independent experiments. bPRL: bMEC treated with bovine prolactin (5 ng/mL). The symbol ‘‘*’’ indicates significant changes (P < 0.05) in relation to control non-infected bMEC. The symbol ‘‘**’’ indicates significant changes (P < 0.05) in relation to the same condition but non-infected bMEC (control or with bPRL).
analysis of cytokines TNF-a and IL-1b. In agreement with Strandberg et al. (2005) and Boutet et al. (2007), bPRL (5 ng/mL) promoted a slightly up-regulation of TNF-a in bMEC non-infected, whereas S. aureus infection significantly enhanced the expression of TNF-a. Interestingly, the infection and bPRL together
reduced IL-1b mRNA and did not change TNF-a (Fig. 3). To our knowledge, the present findings represent the first evidence that bPRL and S. aureus infection down-regulate the expression for IL-1b in bMEC. In vitro studies using endothelial cells and the immortal cell line MAC-T indicate that the infection
Table 2 NO production by bMEC
Control bMEC infected with Staphylococcus aureus a b c
Without bPRL (mM S.E.M.)a
With 5 ng/mL bPRL (mM S.E.M.)
0.4111 0.0001 4.9526 0.792c
1.3799 0.31b 4.0443 0.0004b
Values are means S.E.M. of three independent experiments of NO secreted by bMEC, measured as nitrite concentration. Significantly different (P < 0.05) from the respective value without bPRL, as calculated by Student’s t-test. Significantly different (P < 0.05) from value for non-infected cells, as calculated by Student’s t-test.
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with S. aureus induced apoptosis (Menzies and Kourteva, 1998). Preliminary results of our laboratory demonstrated that bMEC start dying after 8 h of infection with S. aureus, but further experiments are necessary to determine if apoptosis is induced in our model. Under inflammatory conditions, bMEC are able to produce NO in mammary gland (Onona and Inano, 1998; Boulanger et al., 2001). Additionally, PRL modulates the expression of iNOS in fibroblasts (Corbacho et al., 2003). In agreement with these results, we detected an induction of iNOS mRNA in bMEC treated with bPRL and under infection conditions (Fig. 4d). In general, these results have correlation with NO production in bMEC. Thus, this is the first report that shows an induction for iNOS expression upon S. aureus infection in bMEC. Likewise, in addition to the effect of bPRL on iNOS mRNA expression, this hormone can be regulating other mechanisms necessary for NO production, for example Ca2+-dependent iNOS activation, as has been reported in mouse mammary epithelium (Bolander, 2002, 2005), which could explain the down-regulation in NO production in bMEC infected and treated with bPRL. The expression of b-defensin upon inflammatory conditions has been reported in bovine mammary epithelial cells (Strandberg et al., 2005), but its expression regulated by lactogenic hormones remains unknown. bMEC only treated with bPRL (5 ng/mL) and under infection conditions together with bPRL showed a down-regulated expression of b-defensin mRNA (Fig. 4b). On the other hand, bMEC only infected showed an increase in the expression of b-defensin. These results suggest that bPRL and infection regulate the expression of elements of host innate immune response such as defensins, but the triggered mechanisms remain unknown. Additionally, the k-casein expression was up-regulated by bPRL as well as by infection with S. aureus, suggesting that k-casein mRNA is induced through internalization (Fig. 4e). The expression of caseins in response to the activation of JAK/Stat pathway induced by PRL is well known (Groner, 2002). Additionally, the up-regulation of the JAK/Stat pathway by intracellular bacteria has been reported for the infection with Chlamydia trachomatis (Lad et al., 2005). Accordingly, it has been suggested that during lactation the low activity of NFkB might be important for the high level of expression of casein genes, which are mainly induced by the activation of JAK/Stat pathway (Geymayer and Doppler, 2000; Neville et al., 2002; Sheehy et al., 2004). NFkB activation induced by bPRL has been reported in MAC-
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T cell stimulated during 30 min with bPRL. In this work we treated bMEC with bPRL during 24 h prior to infection, and the hormone was present throughout in order to mimic in vivo conditions. Further research is necessary to determine if NFkB and JAK2/Stat5 signal transduction pathway are activated or repressed under the experimental conditions showed in this work. In conclusion, bPRL stimulates the internalization of viable S. aureus into bovine mammary epithelial cells. These cells were able to raise different innate immune responses to infection in the presence of the hormone, suggesting that bPRL together with S. aureus regulate diverse host innate immune elements. These results may help to explain the reduced innate immune response detected in cows with chronic mastitis attributable mainly to S. aureus (Lee et al., 2006). However, the role of bPRL in cows with coliform mastitis needs further investigation because during in vivo infections, cows challenged with Escherichia coli and S. aureus show different innate immune responses (Bannerman et al., 2004). Acknowledgments We thank Luz Torner-Aguilar for careful reading and helpful suggestions of the manuscript. J.L.A.L. and P.D.L.L. were supported by scholarships from CONACyT. This work was supported by grants from Coordinacio´n de la Investigacio´n Cientı´fica (UMSNH, CIC 14.1) and CONACyT (46400) to A.O.Z. and UMSNH, CIC 14.5 to J.E.L.M. References Alluwaimi, A.M., 2004. The cytokines of bovine mammary gland: prospects for diagnosis and therapy. Res. Vet. Sci. 77, 211–222. Anaya-Lo´pez, J.L., Contreras-Guzma´n, O.E., Ca´rabez-Trejo, A., Baizabal-Aguirre, V.M., Lo´pez-Meza, J.E., Valdez-Alarco´n, J.J., Ochoa-Zarzosa, A., 2006. Invasive potential of bacterial isolates associated with subclinical bovine mastitis. Res. Vet. Sci. 81, 358–361. Bannerman, D.D., Paape, M.J., Lee, J.W., Zhao, X., Hope, J.C., Rainard, P., 2004. Escherichia coli and Staphylococcus aureus elicit differential innate immune responses following intramammary infection. Clin. Diagn. Lab. Immunol. 11, 463–472. Bolander, F.F., 2001. The role of nitric oxide in the biological activity of prolactin in the mouse mammary gland. Mol. Cell. Endocrinol. 174, 91–98. Bolander, F.F., 2002. Prolactin activation of mammary nitric oxide synthase: molecular mechanisms. J. Mol. Endocrinol. 28, 45–51. Bolander, F.F., 2005. The compartmentalization of prolactin signaling in the mouse mammary gland. Mol. Cell. Endocrinol. 245, 105–110.
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