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Effects of fructooligosaccharide-inulin on Salmonella-killing and inflammatory gene expression in chicken macrophages
Veterinary Immunology and Immunopathology 149 (2012) 92–96
Contents lists available at SciVerse ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Short communication
Effects of fructooligosaccharide-inulin on Salmonella-killing and inflammatory gene expression in chicken macrophages Uma S. Babu ∗ , Katelyn Sommers, Lisa M. Harrison, Kannan V. Balan Immunobiology Branch, Food and Drug Administration, Laurel, MD 20708, USA
a r t i c l e
i n f o
Article history: Received 10 January 2012 Received in revised form 12 April 2012 Accepted 2 May 2012 Keywords: Prebiotics Chicken macrophage cells Salmonella Enteritidis
a b s t r a c t Salmonella Enteritidis (SE) is one of the leading causes of food-borne salmonellosis, and macrophages play an essential role in eliminating this pathogen. Among the interventions to improve Salmonella clearance in chickens are the use of prebiotics and direct fed microbials (DFM) in animal feed as they have immunomodulatory effects. Therefore, we tested the influence of a prebiotic fructooligosaccharide (FOS)-inulin on the ability of the chicken macrophage HD11 cell line to phagocytose and kill SE, and express selected inflammatory cytokines and chemokines in an in vitro model. There were significantly fewer viable intracellular SE in HD11 cells treated with FOS-inulin than the untreated cells. However, SE phagocytosis, nitric oxide expression or production were not influenced by the prebiotic treatment. Among the inflammatory markers tested, IL-1 expression was significantly lower in HD11 cells treated with FOS-inulin. These results suggest that FOS-inulin has the ability to modulate the innate immune system as shown by the enhanced killing of SE and decreased inflammasome activation. Published by Elsevier B.V.
1. Introduction Salmonella enterica serovars Enteritidis (SE) and Typhimurium (ST) are facultative intracellular pathogens that may cause serious illness in poultry and humans. Human infections by these bacteria typically occur via food-borne transmission. SE infection is usually associated with the consumption of raw or undercooked contaminated eggs and ST is transmitted by contaminated chicken meat (Holtby et al., 2006; Little et al., 2007; McPherson et al., 2006). For food animals, therapeutic antibiotics are used to control economically important infections. However, with recent concerns of bacterial antibiotic resistance and the presence of antibiotic residues in meat, alternative methods such as dietary interventions are being evaluated to reduce or eliminate Salmonella
∗ Corresponding author at: FDA, MOD I, 8301 Muirkirk Rd., Laurel, MD 20708, USA. Tel.: +1 301 210 7503; fax: +1 301 210 4769. E-mail address: [email protected] (U.S. Babu). 0165-2427/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.vetimm.2012.05.003
colonization in chickens (Babu and Raybourne, 2008). Notable among the interventions are the use of prebiotics and direct fed microbials (DFM) in animal feed as they have shown to have immunomodulatory effects by boosting the host immune response and thus resulting in resistance to infections. Macrophages represent the first-line of defense and mediate crucial innate immune responses to bacterial infections. Upon infection, Salmonella invades host macrophages and can induce cell death or establish intracellular survival within phagocytic vacuoles. In a mouse model of ST, macrophage cell death is a result of Caspase-1dependent activation of pro-inflammatory cytokines IL-1ß and IL-18 (Monack et al., 2001). Persistent survival within macrophages (Babu et al., 2006; Wigley et al., 2001) is attributed to the Salmonella pathogenicity island 2 (SPI 2), which is known to inhibit NADPH oxidase-dependent killing of the bacteria (Vazquez-Torres et al., 2000), resulting in dissemination of Salmonella to the spleen, liver, intestinal and reproductive tissues (Henderson et al., 1999; Okamura et al., 2001).
U.S. Babu et al. / Veterinary Immunology and Immunopathology 149 (2012) 92–96
Prebiotics are non-viable and non-digestible food components that confer a health benefit to the host through modulation of the large intestinal microbiota. In addition, prebiotic effects may influence the immune system directly or indirectly as a result of intestinal fermentation and alteration of gut microbiota (Roberfroid et al., 2010). Fructooligosaccharide products (FOS, oligofructose and inulin) and mannanoligosaccharides are the most commonly used oligosaccharides in chicken either individually or in combination with DFMs for their activity against Salmonella colonization (Agunos et al., 2007; Bailey et al., 1991; Chambers et al., 1997; Fernandez et al., 2002). In an in vitro study, -1,4 mannobiose-treated chicken macrophages showed increased Salmonella-killing activity. Moreover, in the absence of Salmonella, -1,4 mannobiose treatment resulted in expression of genes involved in antimicrobial and innate host defense mechanisms (Ibuki et al., 2011). The objectives of this study were to assess the role of FOS-inulin on the functional activity of chicken macrophages including phagocytosis, their ability to kill SE, production of nitric oxide (NO) and expression of proinflammatory cytokines. 2. Materials and methods 2.1. Cell line and prebiotic treatment Chicken macrophage-like cells, HD11, were generously provided by Dr. Hang Xie (USDA, Beltsville, MD). The cells were maintained in RPMI 1640 tissue culture medium (Hyclone, Logan, UT) supplemented with 8% heatinactivated chicken (Sigma-Aldrich, St. Louis, MO) and 4% heat-inactivated fetal bovine (Hyclone) serum, antibiotics, glutamine and non-essential amino acids. For infection experiments, cells from overnight culture flasks (41 ◦ C, 5% CO2 ) were washed twice with RPMI 1640 tissue culture medium without serum and antibiotics and re-suspended in the same medium at a concentration of 1 × 106 cells/ml. Cells were starved in this serum free medium for 2 h and then switched to antibiotic free serum medium containing 100 g/ml prebiotic Orafti Synergy1 (Beneo Inc., Morris Plains, NJ), which is fructooligosaccharide-enriched with inulin (FOS-inulin). Cells were incubated with prebiotics for 5 h prior to SE infection. Previously, doses ranging from 50 to 200 g/ml FOS-inulin were evaluated for macrophage toxicity, and were found to be safe up to 200 g/ml. 2.2. In vitro infection of macrophages Green fluorescent protein (GFP)-labeled SE was developed and maintained as previously described (Xie et al., 2003). Logarithmic growth phase cultures in Luria broth (37 ◦ C) were washed in sterile phosphate buffered saline (PBS) and adjusted to an OD550 of 0.4. This provided an approximate count of 108 colony forming units (CFU)/ml. In vitro macrophage infection and flow cytometric evaluation of infected macrophages were carried out as previously described (Xie et al., 2003). Briefly, bacteria were added to 1 ml of 106 prebiotic-incubated or untreated macrophage suspensions in tubes (in RPMI without antibiotics) at a ratio of 50–100 bacteria:1 macrophage. The infected
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suspensions were incubated at 37 ◦ C and in 5% CO2 atmosphere for one hour with gentle shaking. Upon initial uptake, cells were washed and resuspended in complete RPMI 1640 medium containing gentamicin (50 g/ml) (GIBCO, Grand Island, NY) to kill extracellular SE. After 1 h incubation in this medium, cells were washed and resuspended in complete medium containing gentamicin at 10 g/ml and incubated overnight at 37 ◦ C/5% CO2 . At 12–16 h post infection, cells were analyzed for bacterial uptake by flow cytometry. Bacterial killing by macrophages was assessed by plating 100 l cell lysates on trypticase soy agar and enumerating the colony forming units (CFU). Remaining cells were used for gene expression analyses and cell supernatants were tested for nitric oxide (NO) production.
2.3. Nitric oxide analysis Supernatants (50 l aliquots) from the infected cells were mixed with 50 l Griess reagent (Sigma, St. Louis, MO) and incubated at room temperature for 10 min and were read in an ELISA plate reader (Molecular Devices, Sunnyvale, CA) at 550 nm. The amount of NO produced was calculated by comparing with a standard curve produced by using 0–200 nmoles of NaNO2 . 2.4. RNA extraction and real-time RT-PCR for gene expression Cells were lysed with QIAzol (Qiagen, Valencia, CA) and RNA was separated in the aqueous phase by adding chloroform. RNA was then purified by EZ1 Advanced XL extractor according to the manufacturer’s instructions (Qiagen). The purified RNA was treated with DNase I to remove genomic DNA contamination prior to cDNA preparation using a RT2 First-strand kit (Qiagen), and 20 ng cDNA was used for each real-time SYBR Green-based PCR reaction. PCR amplification for IL-1, LITAF, CCL4 and iNOS were carried out at 55 ◦ C (30 s/40 cycles) on a CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA) using pre-validated primers (Qiagen). Since SYBR Green I dye binds non-specifically to any double-stranded DNA, a dissociation or melting reaction was carried out after every amplification reaction to determine the specificity of the amplified product without gel electrophoresis or sequencing. Relative gene expression was calculated by using the web-based RT2 Profiler PCR data analysis software (ver. 3.4, Qiagen) using GAPDH as an internal control. Gene expression in SE-infected but untreated cells were normalized to 1 and the changes observed in the prebiotic-treated SEinfected cells are presented.
2.5. Statistical analysis Data pertaining to SE killing and NO production were analyzed by T test using Sigma Stat software v3.5 (Systat Software Inc, San Jose, CA). Differences were considered statistically significant at p ≤ 0.05. For gene expression data, p values were calculated based on a Student’s t-test of the
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Fig. 1. Representative histograms of Salmonella phagocytosis by HD11 cells in the absence (A) and presence (B) of FOS. HD11 cells were pre-treated for approximately 5 h with FOS (100 g/ml) and then infected with GFP-SE for 2 h, washed and incubated overnight in RPMI with gentamicin (10 g/ml). These cells were analyzed by flow cytometry (1000 cells/sample) for bacterial uptake. The x-axis of each histogram is relative GFP fluorescence (log scale) and the y axis is number of cells. Marker regions show the peak positive for GFP with the percentage of cells in that region shown in the text boxes of each graph (65.3% for SE without FOS and 68.1% for SE with FOS, respectively).
replicate 2ˆ(- Delta Ct) values for each gene and differences are considered significant at p ≤ 0.05.
3. Results and discussion The interaction between Salmonella and phagocytic cells such as macrophages and heterophils constitutes the first line of host defense, which includes production of reactive oxygen and nitrogen species, inflammatory cytokines, chemokines and a host of antibacterial enzymes and chemicals that can destroy the intracellular pathogen (Flannagan et al., 2009; Mastroeni, 2002; Tam et al., 2008). The role of macrophages in Salmonella defense is implied by an increased macrophage population in the reproductive tissues of laying hens infected with SE (Withanage et al., 2003). For more than a decade it has been demonstrated that fructooligosaccharide alone or in combination with the competitive exclusion cultures can decrease organ colonization and recovery of SE from the cecal contents of young white Leghorn chickens and broiler chicks (Bailey et al., 1991; Fukata et al., 1999). In addition, a recent in vitro study showed a dose and time dependent increase in SE clearance by pre-treating a chicken macrophage cell line (MQ-NCSU) with the prebiotic 1-4 mannobiose (Ibuki et al., 2011). Our study shows that phagocytosis of SE by HD11 cells was not affected by FOS-inulin pretreatment (Fig. 1). However, the prebiotic treatment caused a significant increase in the clearance of SE by HD11 cells (Fig. 2). Among the antimicrobial mechanisms of phagocytic cells of murine and avian species, oxidative and nitroasive responses are known to play an important role (VazquezTorres and Fang, 2001; Withanage et al., 2005a). Our previous work has shown that SE infection caused a significant increase in NO production by HD11 cells with little or no superoxide generation (Babu et al., 2006). In this study we observed a significant increase in NO production by SE infected HD11 cells compared to the uninfected controls accompanied by higher iNOS expression (data not shown). However, prebiotic treatment did not influence the NO production thus suggesting that the FOS-inulin-mediated bacterial clearance was not mediated by NO.
Salmonella infection has been shown to upregulate many inflammatory genes in the tissues of susceptible chickens and in avian cell systems (Chappell et al., 2009; Cheeseman et al., 2008; Ciraci et al., 2010; van Hemert et al., 2006). Similarly, we observed an increased expression of IL-1, LITAF, CCL4 and iNOS in SE-infected HD11 cells (data not shown). However, expression of IL-1 was significantly reduced in SE-infected HD11 cells following FOS-inulin treatment (Fig. 3). In a mouse model it has been demonstrated that invasion of macrophages by Salmonella Typhimurium causes caspase-1-dependent cell death leading to activation of the potent pro-inflammatory cytokines IL-1 and IL-18, which is necessary for efficient colonization of the mouse gastrointestinal tract (Monack et al., 2001). Various immune and inflammatory mediators have been shown to interact in a complex network to mount a protective immune response to Salmonella (Ciraci et al., 2010). Although these cytokines and chemokines generated by phagocytic cells are intended for protection against pathogens, some of the inflammatory mediators such as IL-1 and IL-8 may have a detrimental effect as increased levels of these cytokines in cecal tonsils have been associated with increased tissue colonization in Salmonella susceptible chickens compared to the resistant
Fig. 2. Intracellular SE within HD11 cells after 24 h of infection without and with FOS. HD11 cells were pre-treated for approximately 5 h with FOS (100 g/ml) and then infected with GFP-SE for 2 h, washed and incubated overnight in RPMI with gentamicin (10 g/ml). These cells were diluted, lysed and plated on trypticase soy agar plates for colony enumeration. Each bar represents mean ± SEM from 8 to 10 replicates. Different superscripts on the two bars indicate a significant difference at p < 0.05.
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dietary polysaccharide -glucan had no impact on splenic MIP-1 (CCL4) in any of these chicken lines. Furthermore, it was demonstrated that the Fayoumi line was more sensitive to diet-induced changes on splenic IL-4, IL-6 and IL-18 than the outbred broilers or Leghorn chickens (Redmond et al., 2010). Overall, our results suggested that prebiotic FOS-inulin improved the ability of HD11 cells to clear Salmonella Enteritidis by preventing IL-1- associated macrophage cell death. However, the efficacy of FOS-inulin as a dietary intervention to reduce or eliminate Salmonella colonization in laying hens and thereby prevent egg contamination has to be validated by conducting in vivo studies preferably in field settings. Fig. 3. Effect of FOS on the expression of IL-1, LITAF, CCL4 and iNOS genes in HD11 cells with FOS pretreatment. HD11 cells were pre-treated for approximately 5 h with FOS (100 g/ml) and then infected with GFP-SE for 2 h, washed and incubated overnight in RPMI with gentamicin (10 g/ml). These cells were then washed with PBS, total RNA was extracted and gene expression was analyzed by real-time RT-PCR as described in the Section 2. Results are expressed as fold change relative to the SE-infected cells without FOS pre-treatment. Data are means of 6–8 independent experiments ± SEM. *Indicates that IL-1 expression was lower in cells pre-treated with FOS at p < 0.05.
This study was supported by the Food and Drug Administration Commissioner’s Fellowship Program and the Oak Ridge Institute for Science and Education.
line (Sadeyen et al., 2004). Furthermore, a direct correlation between increased IL-1 expression and histopathological changes in the ileum and liver of newly hatched chicks infected with Salmonella Typhimurium has been reported previously (Withanage et al., 2004). Therefore, our observation of reduced IL-1 expression in SE-infected HD11 cells pre-treated with FOS-inulin suggests a protective effect of the prebiotic on chicken macrophages. Further experiments need to be conducted to evaluate the causal relationship between IL-1 and SE-mediated macrophage apoptosis by blocking IL-1 with neutralizing antibodies or by adding soluble type-1 IL-1 receptors (Klasing and Peng, 2001). We measured the expression of LITAF, which is reported to be involved in the activation of TNF-␣ expression following LPS stimulation (Myokai et al., 1999). LITAF has been demonstrated to play a protective role in SE infection in the resistant White Leghorn chicken line suggesting that it may play a role in bacterial clearance and initiate signaling cascades involved in innate immune responses (Chausse et al., 2011). In this study we saw an increased expression of LITAF by SE (data not shown), but it was not further enhanced by the prebiotic treatment (Fig. 3). In our study, FOS-inulin did not influence the expression of CCL4 in SE infected HD11 cells (Fig. 3), thus suggesting that the dose or the type of prebiotic used in this study was not effective in modulating CCL4- mediated responses. Likewise, increased expression of CCL4 or macrophage inflammatory protein- 1 (MIP-1) has been reported in the intestinal tissues of newly hatched chickens infected with Salmonella Typhimurium (Withanage et al., 2004). Although CCL4 is an inflammatory mediator, it is believed to attract immune cells to the gut and result in further activation thus playing a protective role in Salmonella resistance (Withanage et al., 2005b). A recent study conducted with diverse chicken lines such as outbred broiler and highly inbred Leghorn and Fayoumi lines showed that
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Acknowledgement
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Contents lists available at SciVerse ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Short communication
Effects of fructooligosaccharide-inulin on Salmonella-killing and inflammatory gene expression in chicken macrophages Uma S. Babu ∗ , Katelyn Sommers, Lisa M. Harrison, Kannan V. Balan Immunobiology Branch, Food and Drug Administration, Laurel, MD 20708, USA
a r t i c l e
i n f o
Article history: Received 10 January 2012 Received in revised form 12 April 2012 Accepted 2 May 2012 Keywords: Prebiotics Chicken macrophage cells Salmonella Enteritidis
a b s t r a c t Salmonella Enteritidis (SE) is one of the leading causes of food-borne salmonellosis, and macrophages play an essential role in eliminating this pathogen. Among the interventions to improve Salmonella clearance in chickens are the use of prebiotics and direct fed microbials (DFM) in animal feed as they have immunomodulatory effects. Therefore, we tested the influence of a prebiotic fructooligosaccharide (FOS)-inulin on the ability of the chicken macrophage HD11 cell line to phagocytose and kill SE, and express selected inflammatory cytokines and chemokines in an in vitro model. There were significantly fewer viable intracellular SE in HD11 cells treated with FOS-inulin than the untreated cells. However, SE phagocytosis, nitric oxide expression or production were not influenced by the prebiotic treatment. Among the inflammatory markers tested, IL-1 expression was significantly lower in HD11 cells treated with FOS-inulin. These results suggest that FOS-inulin has the ability to modulate the innate immune system as shown by the enhanced killing of SE and decreased inflammasome activation. Published by Elsevier B.V.
1. Introduction Salmonella enterica serovars Enteritidis (SE) and Typhimurium (ST) are facultative intracellular pathogens that may cause serious illness in poultry and humans. Human infections by these bacteria typically occur via food-borne transmission. SE infection is usually associated with the consumption of raw or undercooked contaminated eggs and ST is transmitted by contaminated chicken meat (Holtby et al., 2006; Little et al., 2007; McPherson et al., 2006). For food animals, therapeutic antibiotics are used to control economically important infections. However, with recent concerns of bacterial antibiotic resistance and the presence of antibiotic residues in meat, alternative methods such as dietary interventions are being evaluated to reduce or eliminate Salmonella
∗ Corresponding author at: FDA, MOD I, 8301 Muirkirk Rd., Laurel, MD 20708, USA. Tel.: +1 301 210 7503; fax: +1 301 210 4769. E-mail address: [email protected] (U.S. Babu). 0165-2427/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.vetimm.2012.05.003
colonization in chickens (Babu and Raybourne, 2008). Notable among the interventions are the use of prebiotics and direct fed microbials (DFM) in animal feed as they have shown to have immunomodulatory effects by boosting the host immune response and thus resulting in resistance to infections. Macrophages represent the first-line of defense and mediate crucial innate immune responses to bacterial infections. Upon infection, Salmonella invades host macrophages and can induce cell death or establish intracellular survival within phagocytic vacuoles. In a mouse model of ST, macrophage cell death is a result of Caspase-1dependent activation of pro-inflammatory cytokines IL-1ß and IL-18 (Monack et al., 2001). Persistent survival within macrophages (Babu et al., 2006; Wigley et al., 2001) is attributed to the Salmonella pathogenicity island 2 (SPI 2), which is known to inhibit NADPH oxidase-dependent killing of the bacteria (Vazquez-Torres et al., 2000), resulting in dissemination of Salmonella to the spleen, liver, intestinal and reproductive tissues (Henderson et al., 1999; Okamura et al., 2001).
U.S. Babu et al. / Veterinary Immunology and Immunopathology 149 (2012) 92–96
Prebiotics are non-viable and non-digestible food components that confer a health benefit to the host through modulation of the large intestinal microbiota. In addition, prebiotic effects may influence the immune system directly or indirectly as a result of intestinal fermentation and alteration of gut microbiota (Roberfroid et al., 2010). Fructooligosaccharide products (FOS, oligofructose and inulin) and mannanoligosaccharides are the most commonly used oligosaccharides in chicken either individually or in combination with DFMs for their activity against Salmonella colonization (Agunos et al., 2007; Bailey et al., 1991; Chambers et al., 1997; Fernandez et al., 2002). In an in vitro study, -1,4 mannobiose-treated chicken macrophages showed increased Salmonella-killing activity. Moreover, in the absence of Salmonella, -1,4 mannobiose treatment resulted in expression of genes involved in antimicrobial and innate host defense mechanisms (Ibuki et al., 2011). The objectives of this study were to assess the role of FOS-inulin on the functional activity of chicken macrophages including phagocytosis, their ability to kill SE, production of nitric oxide (NO) and expression of proinflammatory cytokines. 2. Materials and methods 2.1. Cell line and prebiotic treatment Chicken macrophage-like cells, HD11, were generously provided by Dr. Hang Xie (USDA, Beltsville, MD). The cells were maintained in RPMI 1640 tissue culture medium (Hyclone, Logan, UT) supplemented with 8% heatinactivated chicken (Sigma-Aldrich, St. Louis, MO) and 4% heat-inactivated fetal bovine (Hyclone) serum, antibiotics, glutamine and non-essential amino acids. For infection experiments, cells from overnight culture flasks (41 ◦ C, 5% CO2 ) were washed twice with RPMI 1640 tissue culture medium without serum and antibiotics and re-suspended in the same medium at a concentration of 1 × 106 cells/ml. Cells were starved in this serum free medium for 2 h and then switched to antibiotic free serum medium containing 100 g/ml prebiotic Orafti Synergy1 (Beneo Inc., Morris Plains, NJ), which is fructooligosaccharide-enriched with inulin (FOS-inulin). Cells were incubated with prebiotics for 5 h prior to SE infection. Previously, doses ranging from 50 to 200 g/ml FOS-inulin were evaluated for macrophage toxicity, and were found to be safe up to 200 g/ml. 2.2. In vitro infection of macrophages Green fluorescent protein (GFP)-labeled SE was developed and maintained as previously described (Xie et al., 2003). Logarithmic growth phase cultures in Luria broth (37 ◦ C) were washed in sterile phosphate buffered saline (PBS) and adjusted to an OD550 of 0.4. This provided an approximate count of 108 colony forming units (CFU)/ml. In vitro macrophage infection and flow cytometric evaluation of infected macrophages were carried out as previously described (Xie et al., 2003). Briefly, bacteria were added to 1 ml of 106 prebiotic-incubated or untreated macrophage suspensions in tubes (in RPMI without antibiotics) at a ratio of 50–100 bacteria:1 macrophage. The infected
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suspensions were incubated at 37 ◦ C and in 5% CO2 atmosphere for one hour with gentle shaking. Upon initial uptake, cells were washed and resuspended in complete RPMI 1640 medium containing gentamicin (50 g/ml) (GIBCO, Grand Island, NY) to kill extracellular SE. After 1 h incubation in this medium, cells were washed and resuspended in complete medium containing gentamicin at 10 g/ml and incubated overnight at 37 ◦ C/5% CO2 . At 12–16 h post infection, cells were analyzed for bacterial uptake by flow cytometry. Bacterial killing by macrophages was assessed by plating 100 l cell lysates on trypticase soy agar and enumerating the colony forming units (CFU). Remaining cells were used for gene expression analyses and cell supernatants were tested for nitric oxide (NO) production.
2.3. Nitric oxide analysis Supernatants (50 l aliquots) from the infected cells were mixed with 50 l Griess reagent (Sigma, St. Louis, MO) and incubated at room temperature for 10 min and were read in an ELISA plate reader (Molecular Devices, Sunnyvale, CA) at 550 nm. The amount of NO produced was calculated by comparing with a standard curve produced by using 0–200 nmoles of NaNO2 . 2.4. RNA extraction and real-time RT-PCR for gene expression Cells were lysed with QIAzol (Qiagen, Valencia, CA) and RNA was separated in the aqueous phase by adding chloroform. RNA was then purified by EZ1 Advanced XL extractor according to the manufacturer’s instructions (Qiagen). The purified RNA was treated with DNase I to remove genomic DNA contamination prior to cDNA preparation using a RT2 First-strand kit (Qiagen), and 20 ng cDNA was used for each real-time SYBR Green-based PCR reaction. PCR amplification for IL-1, LITAF, CCL4 and iNOS were carried out at 55 ◦ C (30 s/40 cycles) on a CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA) using pre-validated primers (Qiagen). Since SYBR Green I dye binds non-specifically to any double-stranded DNA, a dissociation or melting reaction was carried out after every amplification reaction to determine the specificity of the amplified product without gel electrophoresis or sequencing. Relative gene expression was calculated by using the web-based RT2 Profiler PCR data analysis software (ver. 3.4, Qiagen) using GAPDH as an internal control. Gene expression in SE-infected but untreated cells were normalized to 1 and the changes observed in the prebiotic-treated SEinfected cells are presented.
2.5. Statistical analysis Data pertaining to SE killing and NO production were analyzed by T test using Sigma Stat software v3.5 (Systat Software Inc, San Jose, CA). Differences were considered statistically significant at p ≤ 0.05. For gene expression data, p values were calculated based on a Student’s t-test of the
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Fig. 1. Representative histograms of Salmonella phagocytosis by HD11 cells in the absence (A) and presence (B) of FOS. HD11 cells were pre-treated for approximately 5 h with FOS (100 g/ml) and then infected with GFP-SE for 2 h, washed and incubated overnight in RPMI with gentamicin (10 g/ml). These cells were analyzed by flow cytometry (1000 cells/sample) for bacterial uptake. The x-axis of each histogram is relative GFP fluorescence (log scale) and the y axis is number of cells. Marker regions show the peak positive for GFP with the percentage of cells in that region shown in the text boxes of each graph (65.3% for SE without FOS and 68.1% for SE with FOS, respectively).
replicate 2ˆ(- Delta Ct) values for each gene and differences are considered significant at p ≤ 0.05.
3. Results and discussion The interaction between Salmonella and phagocytic cells such as macrophages and heterophils constitutes the first line of host defense, which includes production of reactive oxygen and nitrogen species, inflammatory cytokines, chemokines and a host of antibacterial enzymes and chemicals that can destroy the intracellular pathogen (Flannagan et al., 2009; Mastroeni, 2002; Tam et al., 2008). The role of macrophages in Salmonella defense is implied by an increased macrophage population in the reproductive tissues of laying hens infected with SE (Withanage et al., 2003). For more than a decade it has been demonstrated that fructooligosaccharide alone or in combination with the competitive exclusion cultures can decrease organ colonization and recovery of SE from the cecal contents of young white Leghorn chickens and broiler chicks (Bailey et al., 1991; Fukata et al., 1999). In addition, a recent in vitro study showed a dose and time dependent increase in SE clearance by pre-treating a chicken macrophage cell line (MQ-NCSU) with the prebiotic 1-4 mannobiose (Ibuki et al., 2011). Our study shows that phagocytosis of SE by HD11 cells was not affected by FOS-inulin pretreatment (Fig. 1). However, the prebiotic treatment caused a significant increase in the clearance of SE by HD11 cells (Fig. 2). Among the antimicrobial mechanisms of phagocytic cells of murine and avian species, oxidative and nitroasive responses are known to play an important role (VazquezTorres and Fang, 2001; Withanage et al., 2005a). Our previous work has shown that SE infection caused a significant increase in NO production by HD11 cells with little or no superoxide generation (Babu et al., 2006). In this study we observed a significant increase in NO production by SE infected HD11 cells compared to the uninfected controls accompanied by higher iNOS expression (data not shown). However, prebiotic treatment did not influence the NO production thus suggesting that the FOS-inulin-mediated bacterial clearance was not mediated by NO.
Salmonella infection has been shown to upregulate many inflammatory genes in the tissues of susceptible chickens and in avian cell systems (Chappell et al., 2009; Cheeseman et al., 2008; Ciraci et al., 2010; van Hemert et al., 2006). Similarly, we observed an increased expression of IL-1, LITAF, CCL4 and iNOS in SE-infected HD11 cells (data not shown). However, expression of IL-1 was significantly reduced in SE-infected HD11 cells following FOS-inulin treatment (Fig. 3). In a mouse model it has been demonstrated that invasion of macrophages by Salmonella Typhimurium causes caspase-1-dependent cell death leading to activation of the potent pro-inflammatory cytokines IL-1 and IL-18, which is necessary for efficient colonization of the mouse gastrointestinal tract (Monack et al., 2001). Various immune and inflammatory mediators have been shown to interact in a complex network to mount a protective immune response to Salmonella (Ciraci et al., 2010). Although these cytokines and chemokines generated by phagocytic cells are intended for protection against pathogens, some of the inflammatory mediators such as IL-1 and IL-8 may have a detrimental effect as increased levels of these cytokines in cecal tonsils have been associated with increased tissue colonization in Salmonella susceptible chickens compared to the resistant
Fig. 2. Intracellular SE within HD11 cells after 24 h of infection without and with FOS. HD11 cells were pre-treated for approximately 5 h with FOS (100 g/ml) and then infected with GFP-SE for 2 h, washed and incubated overnight in RPMI with gentamicin (10 g/ml). These cells were diluted, lysed and plated on trypticase soy agar plates for colony enumeration. Each bar represents mean ± SEM from 8 to 10 replicates. Different superscripts on the two bars indicate a significant difference at p < 0.05.
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dietary polysaccharide -glucan had no impact on splenic MIP-1 (CCL4) in any of these chicken lines. Furthermore, it was demonstrated that the Fayoumi line was more sensitive to diet-induced changes on splenic IL-4, IL-6 and IL-18 than the outbred broilers or Leghorn chickens (Redmond et al., 2010). Overall, our results suggested that prebiotic FOS-inulin improved the ability of HD11 cells to clear Salmonella Enteritidis by preventing IL-1- associated macrophage cell death. However, the efficacy of FOS-inulin as a dietary intervention to reduce or eliminate Salmonella colonization in laying hens and thereby prevent egg contamination has to be validated by conducting in vivo studies preferably in field settings. Fig. 3. Effect of FOS on the expression of IL-1, LITAF, CCL4 and iNOS genes in HD11 cells with FOS pretreatment. HD11 cells were pre-treated for approximately 5 h with FOS (100 g/ml) and then infected with GFP-SE for 2 h, washed and incubated overnight in RPMI with gentamicin (10 g/ml). These cells were then washed with PBS, total RNA was extracted and gene expression was analyzed by real-time RT-PCR as described in the Section 2. Results are expressed as fold change relative to the SE-infected cells without FOS pre-treatment. Data are means of 6–8 independent experiments ± SEM. *Indicates that IL-1 expression was lower in cells pre-treated with FOS at p < 0.05.
This study was supported by the Food and Drug Administration Commissioner’s Fellowship Program and the Oak Ridge Institute for Science and Education.
line (Sadeyen et al., 2004). Furthermore, a direct correlation between increased IL-1 expression and histopathological changes in the ileum and liver of newly hatched chicks infected with Salmonella Typhimurium has been reported previously (Withanage et al., 2004). Therefore, our observation of reduced IL-1 expression in SE-infected HD11 cells pre-treated with FOS-inulin suggests a protective effect of the prebiotic on chicken macrophages. Further experiments need to be conducted to evaluate the causal relationship between IL-1 and SE-mediated macrophage apoptosis by blocking IL-1 with neutralizing antibodies or by adding soluble type-1 IL-1 receptors (Klasing and Peng, 2001). We measured the expression of LITAF, which is reported to be involved in the activation of TNF-␣ expression following LPS stimulation (Myokai et al., 1999). LITAF has been demonstrated to play a protective role in SE infection in the resistant White Leghorn chicken line suggesting that it may play a role in bacterial clearance and initiate signaling cascades involved in innate immune responses (Chausse et al., 2011). In this study we saw an increased expression of LITAF by SE (data not shown), but it was not further enhanced by the prebiotic treatment (Fig. 3). In our study, FOS-inulin did not influence the expression of CCL4 in SE infected HD11 cells (Fig. 3), thus suggesting that the dose or the type of prebiotic used in this study was not effective in modulating CCL4- mediated responses. Likewise, increased expression of CCL4 or macrophage inflammatory protein- 1 (MIP-1) has been reported in the intestinal tissues of newly hatched chickens infected with Salmonella Typhimurium (Withanage et al., 2004). Although CCL4 is an inflammatory mediator, it is believed to attract immune cells to the gut and result in further activation thus playing a protective role in Salmonella resistance (Withanage et al., 2005b). A recent study conducted with diverse chicken lines such as outbred broiler and highly inbred Leghorn and Fayoumi lines showed that
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Acknowledgement
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