Biometals (2014) 27:843–856 DOI 10.1007/s10534-014-9740-9
Lactoferrin differently modulates the inflammatory response in epithelial models mimicking human inflammatory and infectious diseases Alessandra Frioni • Maria Pia Conte • Antimo Cutone • Catia Longhi • Giovanni Musci • Maria Carmela Bonaccorsi di Patti • Tiziana Natalizi Massimiliano Marazzato • Maria Stefania Lepanto • Patrizia Puddu • Rosalba Paesano • Piera Valenti • Francesca Berlutti
•
Received: 30 March 2014 / Accepted: 13 April 2014 / Published online: 26 April 2014 Ó Springer Science+Business Media New York 2014
Abstract Conflicting data are reported on pro- or anti-inflammatory activity of bovine lactoferrin (bLf) in different cell models as phagocytes or epithelial cell lines infected by bacteria. Here we evaluated the bLf effect on epithelial models mimicking two human pathologies characterized by inflammation and infection with specific bacterial species. Primary bronchial epithelium from a cystic fibrosis (CF) patient and differentiated intestinal epithelial cells were infected with Pseudomonas aeruginosa LESB58 isolated from a CF patient and Adherent-Invasive Escherichia coli LF82 isolated from a Crohn’s disease patient. Surprisingly, bLf significantly reduced the intracellular bacterial survival, but differently modulated the inflammatory response. These data lead us to hypothesize that bLf differentially acts depending on the
epithelial model and infecting pathogen. To verify this hypothesis, we explored whether bLf could modulate ferroportin (Fpn), the only known cellular iron exporter from cells, that, by lowering the intracellular iron level, determines a non permissive environment for intracellular pathogens. Here, for the first time, we describe the bLf ability to up-regulate Fpn protein in infected epithelial models. Our data suggest that the mechanism underlying the bLf modulating activity on inflammatory response in epithelial cells is complex and the bLf involvement in modulating cellular iron homeostasis should be taken into account. Keywords Lactoferrin Inflammation Cystic fibrosis Chron’s disease Ferroportin Infection
This paper is dedicated to the memory of our colleague, Dr. Patriza Puddu who recently passed away. A. Frioni M. P. Conte A. Cutone C. Longhi T. Natalizi M. Marazzato M. S. Lepanto P. Valenti F. Berlutti (&) Microbiology Section, Department of Public Health and Infectious Diseases, University Sapienza of Rome, P.le A. Moro 5, 00185 Rome, Italy e-mail:
[email protected]
M. C. B. di Patti Department of Biochemical Sciences, Sapienza University, P.le A. Moro 5, 00185 Rome, Italy
G. Musci Department of Biosciences and Territory, University of Molise, c.da Fonte Lappone, Pesche, IS, Italy
R. Paesano Department of Women’s Health and Territorial Medicine, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
P. Puddu Istituto Superiore di Sanita`, Viale Regina Elena, 299, 00161 Rome, Italy
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Introduction Lactoferrin (Lf), a key factor in the host defense system, belongs to innate non specific immune system (Wiesner and Vilcinskas 2010). Differently to the most antimicrobial proteins or other molecules of the immune system, Lf acts as modulator of the inflammatory process ranging from inhibition to promotion of inflammation. Literature data are crammed with results showing contradictory effects of Lf on inflammatory processes in in vitro different cell models. Of course, these conflicting data makes it difficult to decipher the actual Lf mechanisms through which the molecule acts. However, a deep analysis of conflicting in vitro results reveals the influence of experimental conditions as the use of phagocytic or epithelial cell lines, the different type of infecting agents as bacteria or viruses, or the stimulation with pathogen-associated molecular patterns (PAMPS) as flagellin, toxin, peptidoglycan, lypopolisaccharide (LPS), and, finally, the use of Lf from different sources (Puddu et al. 2007; Hayworth et al. 2009; Latorre et al. 2012; Legrand 2012). As matter of the fact, Lf is able to induce IL-6 production both in murine peritoneal macrophages trough both a TLR-4 dependent and independent pathway (Curran et al. 2006; Puddu et al. 2007) and in freshly isolated blood monocytes (Puddu et al. 2011). Conversely, the ability of Lf to bind LPS limits the interaction of this PAMP with cellular or soluble receptors, thus reducing the intensity of LPS-mediated inflammatory response of phagocytic cells (Puddu et al. 2011; Latorre et al. 2012). Unlike phagocytes, the epithelial cells are more responsive to live bacteria than to their components as LPS (Cario et al. 2000; Vora et al. 2004). The Lf ability in modulating inflammatory response was detectable only when the cell lines were injured using a strong stimulus such as living microorganisms or toxins (Berlutti et al. 2006; Valenti et al. 2011; Legrand 2012; Komatsu et al. 2013). In these models, Lf exerts essentially an antiinflammatory activity thus reducing the inflammatoryrelated cell damage. Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) (Boucher 2002). In airway epithelia, mutated CFTR leads to perturbations in the regulation of several intracellular signaling pathways resulting in excessive
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production of NF-jB-dependent cytokines such as interleukin (IL)-1, IL-6 and IL-8 (Nichols et al. 2007). Moreover, the CFTR dysfunction leads to abnormalities in iron transport across the airway epithelial cells (O’Sullivan and Flume 2009) and to iron homeostasis disorders (Moreau-Marquis et al. 2008; Wang 2010) as demonstrated by the altered expression of the storage protein ferritin (Ft), the iron importer DMT1, and the exporter ferroportin (Fpn) (Ghio et al. 2005). The high iron concentrations in airway secretions (Reid et al. 2004, 2007) precede the introduction of microbes into the respiratory tract favoring their multiplication and contributing to an increased production of reactive oxygen species that in turn enhance the inflammatory status (Khan et al. 1995; Tirouvanziam et al. 2002). Therefore, the airway of CF subjects is inherently prone to infection that begins in very early life (Ranganathan et al. 2011). By adulthood, the chronic airway infection is sustained in the 80 % of cases by Pseudomonas aeruginosa (Tunney et al. 2008). The iron excess in CF airway secretions stimulates P. aeruginosa to adopt the biofilm lifestyle (Lee et al. 2003; Berlutti et al. 2005; Moreau-Marquis et al. 2008). Bacterial biofilm invades CF airway epithelial cells that respond by activating a strong inflammatory response through elevation of IL-6 and IL-1b levels that, in turn, induces cell damage (Moreau-Marquis et al. 2008; Sagel et al. 2009; Valenti et al. 2011). Crohn’s disease (CD) is a chronic inflammatory bowel disease, characterized by a deregulate mucosal immune response to gut microbiota. Immunologically, CD patients produce high amounts of cytokines including interferon (IFN)-c in the inflamed lamina propria as the result of abnormal Th1- and Th17mediated immune responses (Strober and Fuss 2011). Further evidences support the idea that adherentinvasive Escherichia coli (AIEC), a commensal with the potential to cause disease, may have a causative role in initiating and perpetuating the intestinal inflammation in CD patients (Sasaki et al. 2007; Rolhion and Darfeuille-Michaud 2007; MartinezMedina et al. 2009; Chassaing et al. 2013). AIEC strains colonize the ileal epithelium of CD patients and are able to adhere to and invade intestinal epithelial cells and to replicate within macrophages (Glasser et al. 2001; Darfeuille-Michaud et al. 2004). AIEC adhesion/invasion is dependent on the expression of both type 1 pili expression on the bacterial surface and
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of carcinoembryonic antigen–related cell adhesion molecule (CEACAM6) expression on the apical surface of intestinal epithelial cells. Moreover it has been demonstrated that CEACAM6 expression in cultured intestinal epithelial cells is upregulated both by AIEC infection and by stimulation with the proinflammatory cytokine IFN-c (Barnich et al. 2007). Lf levels have been shown to increase dramatically in body fluids during the course of inflammation. In particular, in CF bronchial secretions the high levels of pro-inflammatory cytokines, including IL-8, recruit neutrophils which synthesize and secrete Lf. As a consequence, Lf is found at higher concentrations in airway secretion of CF patients than in healthy humans (up to 0.1 and 0.01 mg/ml, respectively) (Sagel et al. 2009). In CD patients the high levels of Lf found in feces are proportional to the neutrophil flux (Pfefferkorn et al. 2010). Here we evaluate the role of bovine Lf (bLf) in modulating the inflammatory responses in two epithelial cell models: primary human airway epithelium deriving from CF patients and colonic epithelial cells. The epithelia cell models were infected by P. aeruginosa LESB58 and AIEC strain LF82 the two most important bacterial species involved in the pathogenesis of such diseases (Kukavica-Ibrulj et al. 2008; Darfeuille-Michaud et al. 2004). Moreover, since epithelial cells are considered as non-professional phagocytes, we evaluated whether Lf could modulate the expression of ferroportin (Fpn) (Cairo et al. 2011; Recalcati et al. 2012) the only known cellular iron exporter from cells (De Domenico et al. 2007) whose expression is regulated by inflammation (Yang et al. 2002; Ludwiczek et al. 2003; Paesano et al. 2009; Paesano et al. 2010; Cairo et al. 2011) and pivotal in the control of intracellular bacteria multiplication and inflammation (Kasvosve 2013; Nairz et al. 2013).
Materials and methods Bacterial strains Pseudomonas aeruginosa LES58, highly virulent epidemic strain isolated from lung sputum of CF patient (Cheng et al. 1996), and Escherichia coli LF82, isolated from a chronic ileal lesion of a patient with CD, provided by Dr. Arlette Darfeuille-Michaud,
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Universite´ d’Auvergne, France, prototype of adhesive/ invasive E. coli (AIEC) strains, were used. To check purity, the strains were streaked on Tryptose Soy agar (TSA) plates (Oxoid LTD, England) before the experiments. To prepare inoculum, P. aeruginosa LESB58 was grown overnight at 37 °C in Chemically defined medium (CDM) containing 100 lM ferric ions as (Fe)2(SO4)3 (IL100-CDM) (Berlutti et al. 2005). Broth cultures were diluted in CDM containing 1 lM ferric ions as (Fe)2(SO4)3 (Berlutti et al. 2005) to obtain 8.0 ± 0.5 9 107 colony forming units (CFUs)/ml. AIEC strain LF82 was grown in Brain Heart Infusion Broth (BHI, Oxoid, Rome, Italy) overnight at 37 °C. Epithelial models Primary human airway epithelium reconstituted in vitro with cells isolated from CF patients and cultivated on microporous filters (Transwells) of 6.5 mm of diameter, with a pore size of 0.4 lm at an air–liquid interface (MucilAir, Epithelix Sa`rl, Geneva, Switzerland) was used. Each epithelium contained about 4 9 105 cells. The epithelium model was composed of fully differentiated basal, goblet, and ciliated cells covered by mucus and it reproduced certain phenotypic characteristics of the CF epithelium as the production of thick and dense mucus. MucilAirTM Culture Medium (Epithelix Sa`rl) was used to maintain the epithelium during the experiments. Bronchial epithelial CFBE cell line, homozygous for the DF508-CFTR mutation (Cozens et al. 1994) was grown in Eagle’s minimum essential medium (MEM, Euroclone, Milan, Italy) supplemented with 10 % fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin (Euroclone, Milan, Italy) and maintained in a humidified atmosphere of 95 % air and 5 % CO2 at 37 °C. Human epithelial colorectal adenocarcinoma cellline (Caco-2) was grown in Dulbecco’s modified Eagle’s medium (DMEM, Euroclone, Milan, Italy), supplemented with 1 % penicillin/ streptomycin and 10 % fetal calf serum (FCS) and maintained in 5 % CO2 atmosphere with 95 % humidity at 37 °C. Caco-2 cells were seeded at a density of 1 9 104 cells/well in 24-well plates and allowed to differentiate for 15 days before infection. When required the differentiated Caco-2 monolayers were pre-stimulated for 48 h with 50 ng/ml of IFN-c.
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Lactoferrin Highly purified bovine milk derivative Lf (bLf) was kindly provided by Morinaga Milk Industries Co., Ltd. (Tokyo, Japan). The purity of bLf was checked according to Siciliano et al. (1999). Lipopolysaccharide (LPS) contamination of bLf, estimated by Limulus Amebocyte assay (LAL Pyrochrome kit, PBI International, Italy), was equal to 0.5 ± 0.06 ng/mg of bLf. The bLf iron saturation was about 22 %. Before biological assays, bLf was sterilized by filtration (Millipore). Cytotoxic and antibacterial activity of bovine lactoferrin To evaluate bLf toxicity on CF epithelium, a total of 50 ll of bLf solutions containing from 0.1 to 1 mg/ml was added to the apical side of CF epithelium. After 4 h of incubation, the excess of the solutions was removed and the viability of the epithelium was assayed using resazurin (R7017, Sigma Aldrich, Italy) assay as indicated by the CF epithelium supplier. To evaluate bLf toxicity on differentiated Caco-2 monolayers, bLf solutions containing from 0.1 to 1 mg/ml were added to Caco-2 cells and after 4 and 24 h of incubation cell viability was determined by light microscopy after staining with 0.02 % Trypan blu (Sigma, Italy) and by cell counting. To evaluate the antibacterial activity of bLf, 5.0 ± 0.5 9 106 and of 1.0 ± 0.4 9 106 CFUs of both P. aeruginosa LESB58 and E. coli LF82 were treated with serial bLf dilutions (from 1.0 to 0. 05 mg/ml, final concentrations) for 4 and 24 h. After incubation at 37 °C, the number of CFUs was determined. Invasion assay CF bronchial epithelium was infected at the apical side with 50 ll of the inoculum containing about 5.0 ± 0.5 9 106 P. aeruginosa LESB58 bacteria (MOI 1:10) with and without 0.1 mg/ml bLf. After 4 h at 37 °C in CO2 atmosphere, the liquid excess was removed from the apical side of the epithelium to restore the air/liquid interface. The medium contained in the lower part of the transwells was recovered and fresh medium added. The infected epithelium was incubated for 20 h at 37 °C in CO2 atmosphere. After incubation, the epithelium was washed and lysed with
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50 ll of 1 % deoxycholic acid for 5 min at 37 °C. Cell lysates were diluted in sterile saline and plated on TSA agar to count adherent bacteria. Adhesion efficiency values were calculated as the percentage of the ratio between the numbers of adherent versus the inoculum. To determine the number of intracellular bacteria, after 24 h of incubation, the apical side of the epithelium was washed with sterile saline and a solution of amikacine (1 mg/ml) and ceftazydime (1 mg/ml) was added to kill the extracellular bacteria. Preliminary experiments showed that this antibiotic mixture killed 1.0 ± 0.5 9 107 UFCs of P. aeruginosa LESB58 in 1 h. After 1 h of incubation, the epithelium was washed and lysed. Cell lysates were diluted in sterile saline and plated on TSA agar to count live intracellular bacteria. Invasion efficiency values were calculated as the percentage of the ratio between the numbers of intracellular versus both infecting and adhering bacteria. The viability of the epithelium was assayed using resazurin assay as above specified. Uninfected and infected CFBE without or with bLf were used to estimate Fpn protein. CFBE cells were seeded in 6-well plates at a density of 5 9 105 cells/ well, in 2 ml of MEM supplemented with 100 lM penicillin-streptomycin, 2 mM glutamine and 10 % FBS. The plates were incubated for 48 h at 37 °C in an atmosphere of 95 % air and 5 % CO2. After incubation, the cells were washed twice with 5 ml of PBS without calcium and magnesium (Sigma Aldrich), and further incubated for 2 h in 5 ml of MEM without penicillin-streptomycin and FBS. In infection experiments, CFBE cells were infected with P. aeruginosa LESB 58 at MOI 1:10 with and without 0.1 mg/ml bLf. After 3 h of infection at 37 °C, supernatants were removed and CFBE cells were incubated with 1 mg/ml amikacine and 1 mg/ml ceftazidime for 1 h to kill the extracellular bacteria. After 1 h of incubation, CFBE cells were washed in PBS and fresh medium containing 0.5 mg/ml amikacine, 0.5 mg/ml ceftazidime, and 0.1 mg/ml bLf was added. After incubation for further 20 h, the cells were treated as detailed below (see ‘‘Western blot’’ section). Companion experiments were performed using uninfected CFBE cells. To evaluate invasion, after antibiotic treatment to kill extracellular bacteria, the cells were lysed, cell lysates were diluted in sterile saline and plated on TSA agar to count live intracellular bacteria. Caco-2 differentiated monolayers unstimulated or stimulated with 50 ng/ml IFN-c (Sigma, Italy) for
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48 h at 37 °C before the experiments, were infected with 1.0 ± 0.2 9 106 AIEC LF82 bacteria/ml in culture medium (MOI 1:10) with and without 0.1 mg/ml bLf. After 3 h of infection at 37 °C, supernatants were removed and Caco-2 cells were incubated with 100 lg/ml gentamicin or with 100 lg/ml gentamicin plus 0.1 mg/ml bLf for 1 h to kill the extracellular bacteria. After the final wash in PBS, Caco-2 differentiated monolayers were lysed by adding 0.5 ml of deionized water containing 0.1 % (vol/vol) Triton X-100. Cell lysates were diluted in sterile saline and plated on TSA agar to count live intracellular bacteria. E. coli LF82 strain was considered invasive when the mean invasion level was superior or equal to 0.1 % of the original inoculum. To evaluate intracellular bacterial survival, infected Caco-2 monolayers were incubated for further 20 h with 50 lg/ml gentamicin without or with 0.1 mg/ml bLf. Survival efficiency was quantified as change (fold) in CFUs at 24 h relative to 4 h. The viability of Caco-2 cells was determined by light microscopy after staining with 0.02 % Trypan blu (Sigma, Italy) and by cell counting at 4 h and at 24 h post-infection.
(1:10,000) and monoclonal anti-actin (BD) (1:10,000). After incubation with the appropriate secondary HRP-conjugated antibody, blots were developed with ECL Prime (GE Healthcare). Ferroportin levels were normalized on actin by densitometry analysis, performed with ImageJ.
Cytokine production
Preliminary experiments were carried out to establish the non-cytotoxic and non-bactericidal concentration of bLf. We found that a 0.1 mg/ml concentration did not affect the bacterial viability nor it exerted a cytotoxic activity, therefore this concentration was used. The P. aeruginosa ability to adhere to and invade CF bronchial epithelium through the apical side was assessed. P. aeruginosa was able to adhere to the apical side of CF bronchial epithelium as 3.3 ± 1.4 9 105 CFUs were found at 24 h. When bLf was added, a reduction, of adherent bacteria (2.3 ± 1.4 9 105 vs 3.3 ± 1.4 9 105 CFUs) was noticed although it was not significant (Fig. 1) (P [ 0.05). P. aeruginosa invaded the CF bronchial epithelium and the addition of bLf at the moment of the infection reduced the P. aeruginosa invasion efficiency of about 50 % (P \ 0.05) (Fig. 1). No significant differences of the epithelium viability after 4 h and at 24 h of incubation in all experimental conditions were observed. Concerning the invasive ability of E. coli strain LF82 at 4 h, different efficiencies were noticed in nonstimulated versus IFN-c- stimulated cells (Fig. 2). When the cells were stimulated with IFN-c, a
ELISA were performed to determine cytokine concentrations on culture medium of uninfected and infected epithelial models in the absence or presence of 0.1 mg/ml bLf by using Human IL-8, IL-6, and IL1b ELISA Max Deluxe Sets (BioLegend, San Diego, CA, USA). Western blot A total of 1.0 ± 0.5 9 107 cells was scraped in 1 mL of PBS containing PMSF 1 mM, harvested by centrifugation at 2,5009g for 5 min and stored at -80 °C. Cells were lysed in 300 ll MOPS 25 mM pH 7.4/NaCl 150 mM/Triton 1 % containing PMSF 1 mM, Leupeptin and Pepstatin 2 lM in ice for 1 h. Total protein content of samples was measured by the Bradford assay. For SDS-PAGE 20 lg of total protein were loaded per lane, for Fpn analysis SDS sample buffer containing DTT was added and samples were loaded without heat treatment. For Western blot analysis, primary antibodies used were: monoclonal anti-Fpn 31A5 (Amgen, described in Ross et al. 2012)
Statistical analysis Results are expressed as means ± standard deviations derived from at least three independent experiments. Student t test was used for Fnp expression and to compare adhesion and invasion efficiency between controls and infecting strains. A bilateral Wilcoxon signed rank test was utilized to compare adhesion and invasion efficiency of LF82 in presence and absence of bLf. In all cases, a P value less than or equal to 0.05 was considered statistically significant.
Results Effect of bLf on infection of epithelial models
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Fig. 1 Adhesion and invasion efficiencies of Pseudomonas aeruginosa LESB58 in primary human CF bronchial epithelium at 24 h. Bovine lactoferrin (bLf) (0.1 mg/ml) was added at the moment of infection and maintained during the first 4 h of incubation. a Adhesion percentage values (%) were calculated
as the ratio between the numbers of adherent versus the inoculum; b invasion percentage values (%) were calculated as the ratio between the numbers of intracellular versus both inoculum and adhering bacteria
significant increase in the mean invasion percentage was observed: 0.9 ± 0.47 % versus 0.15 ± 0.10 % (P = 0.002). Addition of bLf during the infection period significantly decreased fivefolds the number of invasive bacteria (mean invasion percentage of 0.03 ± 0.02 % vs 0.15 ± 0.10 %; P = 0.0002), whilst it was not able to significantly inhibit the LF82 invasion efficiency in IFN-c-stimulated cells (0.7 ± 0.37 % vs 0.9 ± 0.47 %). As shown in Fig. 2b, at 24 h LF82 was able to survive inside Caco-2 cells with a mean survival percentage of 76.17 ± 30.90 %. Both IFN-c and bLf treatments significantly reduced the LF82 intracellular survival efficiency: 0.79 ± 0.30 % in IFN-c stimulated cells and 17.68 ± 13.99 % in bLf treated cells versus control 76.17 ± 30.90 %. Considering the low intracellular survival efficiency of bacteria in IFN-c stimulated cells, the effect of bLf was not significant (2.40 ± 1.99 %). Cell viability did not change significantly at 4 h and at 24 h in all experimental conditions.
Effect of bLf on inflammatory response of uninfected and infected epithelial models
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To evaluate the effect of bLf on inflammatory response of epithelial models, the levels of cytokines secreted in the supernatants were assessed by ELISA at 4 and 24 h (Tables 1, 2). Concerning the CF bronchial epithelium (Table 1), IL-8 was produced at high level even in the absence of bacterial infection. After 4 h of P. aeruginosa LESB58 infection an increased production, as compared to the baseline level was observed with a fivefold increase for IL-8 and a tenfold increase for IL6. At 4 h, the addition of bLf did not significantly influence the cytokine expression of uninfected as well as of infected epithelium. When the cytokine levels were examined at 24 h, significant increases for all cytokines were noticed in infected compared to uninfected CF epithelia (IL-6 and IL-1b more than 100 fold; IL-8 about threefold). Addition of bLf did
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Fig. 2 Invasion and survival efficiencies of Escherichia coli LF82 strain in differentiated Caco-2 monolayers. Caco-2 cells were stimulated with IFN-c (50 ng/ml) 48 h before the experiments; bovine lactoferrin (bLf) (0.1 mg/ml) was added at the moment of infection and maintained during the 24 h of
incubation. a Invasion percentage values were calculated as the ratio between the numbers of intracellular bacteria versus inoculum; Panel b: survival efficiency was quantified as change (fold) in CFUs at 24 h relative to 4 h
not influence the cytokine levels of uninfected epithelia, while significantly reduced IL-8, IL-6, and IL-1b in infected epithelia. Differentiated Caco-2 monolayers did not produce IL-6 at detectable level under all experimental conditions. Concerning IL-1b, this cytokine was expressed mostly by IFN-c-stimulated cells and the addition of bLf did not significantly affect the cytokine production (Table 2). Regarding IL-8, the addition of bLf to uninfected monolayers increased the cytokine level only in IFN-c-stimulated cells at 24 h (Table 2). The infection with LF82 induced a higher IL-8 production with respect to controls in all experimental conditions. Addition of bLf to infected cells induced a IL-8 significant increase only in IFN-c-stimulated cells at 4 h.
Effect of bLf on Fpn protein of uninfected and infected epithelial models To evaluate the effect of bLf on Fpn, the Fpn protein on uninfected and infected epithelial models was assessed by Western blot experiments. Concerning CF, CFBE cell line was used since the number of cells in each CF bronchial epithelium transwell was too low. Preliminary experiments showed that the invasion ability at 4 h of P. aeruginosa LESB58 in CFBE with and without bLf was similar to that observed in CF epithelium model. In particular, bLf reduced the invasion efficiency of about 50 % (data not shown). Fpn level in uninfected cells was not affected by the addition of bLf (Fig. 3). P. aeruginosa infection induced a significant increase
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Table 1 Cytokine production by human primary CF bronchial epithelium Treatment
Cytokines (pg/ml) IL-6 (pg/ml) 4h
24 h
IL-1 b (pg/ml)
IL-8 (pg/ml)
4h
4h
24 h
24 h
None (CTRL)
13.77 ± 3.2
218.7 ± 96.8
0.81 ± 0.1
1.2 ± 0.1
493.03 ± 61.2
1,520.4 ± 646.61
bLf (0.1 mg/ml)
16.98 ± 2.8
218.6 ± 213.1
5.01 ± 3.6
0.6 ± 0.05
567.18 ± 44.6
1,852.7 ± 4.0
P. aeruginosa LESB58
179.19 ± 45.2
30,010.6 ± 18,812.1
2.88 ± 1.0
141.3 ± 8.6
2,632.22 ± 320.7
4,550.7 ± 417.2
P. aeruginosa LESB58 plus bLf
206.55 ± 53.1
9,405.6 ± 5,900.2
3.28 ± 0.9
59.9 ± 18.4
2,979.42 ± 169.8
4,318.2 ± 149.9
P values bLf versus CTRL Infection versus CTRL
NS 0.031
NS 0.0237
NS NS
NS 0.0105
NS 0.025
NS 0.0365
Infection with bLf versus without bLf
NS
0.0418
NS
0.0104
NS
0.0265
Cytokine concentrations were assayed in the medium after 4 and 24 h of treatment. Bovine lactoferrin (bLf) (0.1 mg/ml) was added at the time of experiments; for details on infection protocol see ‘‘Materials and methods’’ section NS not significant
of Fpn with respect to control and the addition of bLf further increased the Fpn synthesis. The protein level of Fpn in unstimulated monolayers was the same under all experimental conditions (Fig. 4). In IFN-c stimulated monolayers, the LF82 infection induced a significant reduction of Fpn that was partially, although significantly, restored by the addition of bLf (Fig. 4).
Discussion We have evaluated the role of bLf on human primary CF bronchial epithelium and colonic epithelial cells infected with bacterial species involved in the pathogenesis of CF airway infection and CD, human pathologies characterized by destructive (pathological) inflammation. Firstly, the ability of bLf to affect bacterial entry in epithelial models was assessed. Concerning the CF bronchial epithelium model, P. aeruginosa LESB58 was not able to enter inside the CF epithelium cells in 3 h at variance with what occurs using epithelial cell lines (Berlutti et al. 2008; Valenti et al. 2011). This observation underlines the pivotal importance in the selection of the cellular model that should be as close as possible to the in vivo situation. Therefore, in the reported experiments the infection time was prolonged
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to allow P. aeruginosa to invade the CF epithelium. P. aeruginosa showed both a good adhesion and invasion efficiencies (Fig. 1). In agreement with previous observations (Berlutti et al. 2008), bLf did not affect P. aeruginosa LESB58 adhesion, but significantly reduced the number of intracellular bacteria (Fig. 1). Concerning the intestinal epithelial model, to mimic inflammatory conditions of CD, characterized by a preponderance of Th1 associated cytokines (Strober and Fuss 2011), cell monolayers were stimulated with IFN-c. Different invasion efficiencies were recorded depending on IFN-c stimulation. In fact, IFN-c stimulated monolayers were invaded at significantly higher efficiency with respect to the unstimulated cells (Fig. 2). This result is not surprising, as it has been demonstrated that the ability of LF82 to adhere/invade to Caco-2 cells was significantly increased when the expression of CEACAM6 was induced after IFN-c stimulation (Barnich et al. 2007). The addition of bLf at the moment of infection significantly reduced the AIEC invasion ability in not IFN-c stimulated monolayers, whereas it was ineffective in reducing invasion in IFN-c-stimulated cells. When we examined the survival efficiency after 24 h of infection (Fig. 2), the addition of bLf significantly reduced the number of intracellular bacteria only in unstimulated cells. The IFN-c stimulation induced a more efficient killing activity and the number of
b
NS
NS
Infection with bLf versus without bLf
NS
NS
NS
1.7 ± 0.9 1.45 ± 1.0
1.2 ± 1.0
14 ± 3.8
NS
NS
NS
18.1 ± 9.4 10.7 ± 7.3
1.4 ± 0.8
13.7 ± 8.5
NS
NS
NS
15.7 ± 7.7 35.6 ± 25.6
25.4 ± 12.5
28.0 ± 18.0
b
a
NS
0.0159
0.0,154
1,011.7 ± 490.3 1,333.2 ± 503.1
25.0 ± 1.8
30.9 ± 29.9
4h
Unstimulated
IL-8 (pg/ml)
NS
0.0217
NS
2,876.4 ± 1,376.8 3,748.4 ± 1,045.6
2,783.0 ± 705.2
1,638.5 ± 1,261.2
24 h
0.045865
0.015864
NS
1,675.6 ± 433.7 2,105.8 ± 855.6
43.9 ± 14.6
79.4 ± 27.7
4h
IFN-c stimulated
Bovine lactoferrin (bLf) (0.1 mg/ml) bLf was added at the time of experiments; for details on infection protocol see ‘‘Materials and methods’’ section
Differentiated Caco-2 monolayers were pretreated for 48 h with IFN-c; for details on infection protocol see ‘‘Materials and methods’’ section
NS not significant
Cytokine concentrations were assayed in the medium after 4 and 24 h of treatment
NS
Infection versus CTRL
P values
9.7 ± 8.8 0.9 ± 1.1
1.4 ± 0.9
0.5 ± 0.7
24 h
4h
4h
24 h
IFN-c stimulateda
Unstimulated
IL-1b (pg/ml)
bLf versus CTRL
AIEC LF82 AIEC LF82 plus bLf (0.1 mg/ml)
bLf (0.1 mg/ml)
None (CTRL)
Treatment
Table 2 Cytokine production by differentiated Caco-2 monolayers
NS
0.0155
0.0161
4,801.6 ± 629.8 5,008.7 ± 641.3
4,595.9 ± 500.4
2,548.3 ± 8.3
24 h
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Fig. 3 Western blot and densitometric evaluations of Fpn expression in CFBE bronchial cells at 24 h. Bovine lactoferrin (bLf) (0.1 mg/ml) was added at the moment of infection and maintained during the first 4 h of incubation. Left panel
representative western blot of Fpn and b actin; right panel mean values of densitometric evaluation of Fpn expression normalized on actin
intracellular bacteria was so low that a putative action of bLf was not highlighted. To the best of our knowledge, the reported data showed for the first time the bLf ability to interfere with the intracellular survival of bacterial pathogens. Since it has been shown that Lf is able to modulate the inflammatory response of epithelial cell lines (Berlutti et al. 2006; Valenti et al. 2011), here we evaluated the putative anti-inflammatory activity of bLf in uninfected and infected epithelial models. BLf differently modulated the inflammatory response of the two epithelial models. In particular, in CF bronchial epithelium model, bLf was unable to modulate the inflammatory response of uninfected and infected cells at 4 h (Table 1). At 24 h, the levels of pro-inflammatory cytokines expressed by infected CF epithelium model greatly increased and the addition of bLf significantly reduced the levels of all these cytokines (Table 1). These data confirm the potent anti-inflammatory function of bLf in CF airway models (Valenti et al. 2011).
Regarding the intestinal epithelial model, the only significant inflammatory response was related to IL-8 production (Table 2). In uninfected cells, the bLf addition did not significantly change the IL-8 level at 4 h, whereas surprisingly it significantly increased IL-8 in IFN-c stimulated cells at 24 h. LF82 infection induced significantly increased levels of IL-8 already in the early phase of infection in both IFN-c and in non-stimulated cells, even if at different extent (Table 2) (Sasaki et al. 2007; Huebner et al. 2011). Moreover, in IFN-c stimulated cells, the bLf addition was able to produce a further significant increase of the cytokine production (P = 0.016). This result suggest that in vivo the bLf-induced increase of IL-8 could contribute to the eradication of the acute infection by recruiting neutrophils in infection sites (Alzoghaibi 2005). Overall IFN-c stimulated monolayers were more responsive to bLf and to intracellular bacteria than the non-stimulated cells in the production of IL-8. These results suggest a putative synergic action of
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Fig. 4 Western blot and densitometric evaluations of Fpn expression in differentiated Caco-2 monolayers at 24 h. Caco-2 cells were stimulated with IFN-c (50 ng/ml) 48 h before the experiments; bovine lactoferrin (bLf) (0.1 mg/ml) was added at
the moment of infection and maintained during the 24 h of incubation. Left panel representative western blot of Fpn and b actin; right panel mean values of densitometric evaluation of Fpn expression normalized on actin
IFN-c and bLf in inducing a strong inflammatory response (Welsh et al. 2011). This is not surprising because IFN-c is a specific hallmark of CD in which high levels of lactoferrin in feces has been related to acute phase of the disease (Lewis 2011). In any case, the role of bLf in the inflammatory diseases in which an altered response of innate an acquired immunity play a pivotal role in the pathogenesis (Strober and Fuss 2011; Cohen and Prince 2012) should be further investigated. Taken together, the bLf abilities in reducing the intracellular survival of bacterial pathogens and in differently modulating the inflammatory response of epithelial models, lead us to hypothesize that bLf acts in different way depending on the epithelial model and infecting pathogen. To clarify the mechanism, we explored whether bLf could modulate the Fpn protein whose expression is regulated by inflammation and whose action, by diminishing the intracellular iron level, determines a non permissive environment for
intracellular pathogens (Yang et al. 2002; Ludwiczek et al. 2003; Paesano et al. 2009; Paesano et al. 2010; Cairo et al. 2011; Kasvosve 2013; Nairz et al. 2013). In CF epithelial model P. aeruginosa infection induced the up-regulation of Fpn protein, while in IFN-c stimulated monolayers AIEC infection resulted in decreased expression of Fpn (Figs. 3, 4). When the effect of bLf on Fpn protein was examined, results showed that bLf was ineffective in uninfected cells, while an up-expression of Fpn was observed in both CF and IFN-c stimulated cells (Figs. 3, 4). This is the first time that the bLf influence on Fpn protein in epithelial cells is shown. Although further experiments are needed, the reported data suggest that mechanism underlying the bLf modulating activity on inflammatory response in epithelial cells could be more complex than hypothesized so far (Valenti et al. 2011; Kim et al. 2012) and that a role of bLf in modulating cellular iron homeostasis should be taken into account.
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854 Acknowledgments This work was granted by Italian Cystic Fibrosis Research Foundation (research project FFC#13/2013 adopted by Delegazione FFC di Palermo e di Vittoria Ragusa Catania 2) to FB and by Sapienza University of Rome to PV; thanks are due to Luigi Rosa for his enthusiastic technical assistance.
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