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BT cationic peptides: Small peptides that modulate innate immune responses of chicken heterophils and monocytes
Veterinary Immunology and Immunopathology 145 (2012) 151–158
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Research paper
BT cationic peptides: Small peptides that modulate innate immune responses of chicken heterophils and monocytes Michael H. Kogut a,∗ , Kenneth J. Genovese a , Haiqi He a , Christina L. Swaggerty a , Yi Wei Jiang b a b
Southern Plains Agricultural Research Center, USDA-ARS, College Station, TX 77845, United States Department of Cell Biology & Genetics, University of North Texas Health Sciences Center, 3500 Camp Bowie, Fort Worth, TX 76107, United States
a r t i c l e
i n f o
Article history: Received 9 June 2011 Received in revised form 10 October 2011 Accepted 31 October 2011 Keywords: Antimicrobial peptide Innate immunity Heterophils Monocytes Chickens Priming
a b s t r a c t Neonatal poultry exhibit a transient susceptibility to infectious diseases during the first week of life that stems from inefficient host defense mechanisms. Yet, the initial host immune response to pathogens is a critical determinant of disease resistance and susceptibility. With this context in mind, novel ways to stimulate or modulate the hosts’ natural immune response is emerging as an important area of interest for food animal producers including the poultry industry. Specifically, we have been investigating new modulation strategies tailored around the selective stimulation of the host’s immune system, and particularly rapid acting innate immunity, as an alternative to direct targeting of microbial pathogens. One such approach that we have been investigating is the use of a group of cationic peptides produced by a Gram-positive soil bacterium, Brevibacillus texasporus (BT peptides). We have previously shown that, provided as a feed additive, BT peptides significantly induced a concentration-dependent protection against cecal colonization and extraintestinal colonization by Salmonella enterica serovar Enteritidis (SE). This protection is not the result of direct antibacterial activity of the BT peptides on the SE since the concentrations used were below the minimum inhibitory concentration for SE. We also found that BT are not absorbed in the intestine, but still induce a significant up-regulation in the functional efficiency of peripheral blood heterophils and monocytes. The mechanisms of this immune modulation are unknown. Here, using in vitro models for measuring: (1) leukocyte oxidative burst, (2) changes in leukocyte cytokine and chemokines gene expression profiles, and (3) phosphorylation of the mitogen activated protein kinases (MAPKs) in leukocytes, we evaluated the role of BT peptides as priming mediators for heterophil and monocyte responses at the level of cell function, gene transcription/expression, and cell phosphorylation following stimulation with inflammatory agonists. BT peptides primed both heterophils and monocytes for an increased oxidative burst and up-regulation in transcription of the pro-inflammatory cytokines IL-1 and IL-6 and inflammatory chemokines CXCLi1 and CXCLi2 induced by inflammatory agonists. In addition, BT peptides induced a rapid (10 min) phosphorylation and activation of the extracellular signal-regulated kinase (ERK1/2) and p38 kinase pathways in primary chicken heterophils. Taken together, we conclude that BT peptides, acting through MAPK pathways, enhance leukocyte functional and pro-inflammatory cytokine and chemokine gene transcription activities. These small cationic peptides may prove useful as immune modulators in neonatal poultry. Published by Elsevier B.V.
∗ Corresponding author. Tel.: +1 979 260 3772; fax: +1 979 260 9332. E-mail addresses: [email protected], [email protected] (M.H. Kogut). 0165-2427/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.vetimm.2011.10.023
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1. Introduction Young poultry are most susceptible to invasive pathogens during the first week post-hatch due to a functionally inefficient innate immune response (Lowry et al., 1997; Wells et al., 1998; He et al., 2008). Consequently, researchers, producers, and veterinarians are constantly developing management strategies to protect commercial and private poultry flocks against invading pathogens including Salmonella species. Research efforts in our laboratory have focused on developing immunoprophylactic strategies (Bischoff et al., 2001; Kogut, 2002; Lowry et al., 2005; He et al., 2005; Kogut et al., 2007, 2010) and selective genetics (Swaggerty et al., 2009) that prevent or control intestinal Salmonella organ and intestinal colonization in poultry. Specifically, our research has concentrated on up-regulating the innate immune response in chickens during this immunologically inefficient first week post-hatch. Recently, a novel Gram-positive bacterium Brevibacillus texasporus (ATCC PTA-5854) was isolated that produces BT, a group of structurally related cationic peptides. BT peptides were found to be highly efficacious against a natural outbreak of colibacillosis in broiler chickens based on improved performance and reduced mortality in comparison with unmedicated birds at a level (12 ppm) that was below the minimal inhibition concentration (MIC) for Escherichia coli (Jiang et al., 2005). In vitro, BT displays efficient bactericidal activity against Grampositive bacteria (MIC of 1 ppm), but a reduced efficacy against Gram-negative bacteria (MIC > 20 ppm). Interestingly, orally delivered BT seems to be completely lacking direct antibacterial activities (12 ppm; Jiang et al., 2005). In addition, chickens given BT as a feed additive for the first four days post-hatch provided protection against both cecal colonization and extra-intestinal Salmonella infections in a concentration-dependent manner and induced the up-regulation of peripheral blood heterophils and monocyte functional activities (Kogut et al., 2007, 2010). These data were intriguing because neonatal chickens are highly susceptible to systemic dissemination of enteric Salmonella infections during the first week post-hatch due to the inefficiency of the innate antibacterial defenses (Lowry et al., 1997; Wells et al., 1998). The exact mechanisms of interaction between BT peptides and heterophils and monocytes in chickens have not been elucidated. Therefore, the objective of the present experiments was to determine whether the immune modulating activities of the BT resulted from a direct stimulation of the innate immune cells or were due to a priming effect of the peptides. 2. Materials and methods 2.1. Preparation of aqueous BT peptides B. texasporus E58 cells were grown in 1 l of LB in an air shaker at 37 ◦ C for 3 days. The culture was spun in a clinical centrifuge at 3000 × g for 15 min. The supernatant was collected, and 500 g of ammonium sulfate was added and dissolved. The sample was spun in the clinical centrifuge
at 3000 × g for 15 min. The pellet was dissolved in 200 ml of distilled water. The solution was then boiled for 15 min and then cooled on ice. The sample was filtered with a 0.2m filter (Nalgene). The filtrate was mixed with 0.2 l of chloroform at room temperature for 20 min with a stir bar. The mixture was separated into two phases by centrifugation in the clinical centrifuge at 3000 × g for 15 min. The organic phase was collected and dried in a vacuum evaporator. The dried chloroform extract was dissolved in 2 ml of sterile distilled water. The solution was fractionated on a C18 reverse-phase high-performance liquid chromatography (HPLC) column by using a gradient from 30% solution B to 55% solution B (solution B was 0.075% trifluoroacetic acid in acetonitrile, and solution A was 0.1% trifluoroacetic acid in water). The resulting fractions were dried, dissolved in sterile distilled water, and analyzed with mass spectrometry for peptide identification. Peak fractions containing BT isomers were pooled and analyzed for anti-Staphylococcus aureus activity with a Kirby–Bauer assay to quantify the BT concentration (with MIC = 0.8 g/ml) before being used in the in vitro heterophil priming assays. 2.2. Experimental animals One-day-old Cobb × Ross straight-run broiler chicks were obtained from a local commercial hatchery and were placed on new pine shavings. Birds were provided water and a balanced, unmedicated ration ad libitum. The feed ration contained or exceeded the levels of critical nutrients recommended by the National Research Council (1994). 2.3. Leukocyte isolation Chicken heterophils and peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood as described previously (Kogut et al., 1995). Briefly, peripheral blood from approximately 100 chicks was pooled, mixed with 1% methylcellulose (1:1, v/v), and centrifuged at 25 × g for 15 min. The supernatant was diluted with Ca2+ - and Mg2+ -free Hanks balanced salt solution, carefully layered onto a discontinuous Histopaque gradient (specific gravity 1.077/1.119) in 50 ml conical centrifuge tubes, and centrifuged at 250 × g for 60 min. The peripheral blood mononuclear cell (PBMC) layer at the 1.077/supernatant interface was collected, washed, and resuspended in RPMI-1640. The heterophils, located below the Histopaque 1.077/1.119 interface, were collected, washed, and resuspended in RPMI-1640. Heterophils and PBMC were counted on a hemacytometer and kept on ice until used. Heterophil and PBMC preparations obtained by this method were typically >98% pure and >95% viable. Aliquots of 200 l of PBMC (1 × 107 cells/ml) were dispensed to a 96-well round-bottom plate and incubated at room temperature for 2 h. After incubation, non-adherent cells were removed by washing twice with the culture medium (Dulbecco’s Modified Eagles Medium [DMEM]) containing 10% chicken serum, antibiotics (100 U penicillin/ml and 100 g streptomycin/ml), and 1.5 mM l-glutamine. The adherent monocytes were used for the oxidative burst assay described below.
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Table 1 Real-time quantitative RT-PCR probes and primers. RNA target 28S Probe Fb Rc IL-1 Probe F R IL-6 Probe F R CXCLi1 Probe F R CXCLi2 Probe F R
Probe/primer sequence
Accession numbera
5 -(FAM)-AGGACCGCTACGGACCTCCACCA -(TAMRA)-3 5 -GGCGAAGCCAGAGGAAACT-3 5 -GACGACCGATTGCACGTC-3
X59733
5 -(FAM)-CCACACTGCAGCTGGAGGAAGCC-(TAMRA)-3 5 -GCTCTACATGTCGTGTGTGATGAG-3 , 5 -TGTCGATGTCCCGCATGA-3
AJ245728
5 -(FAM)-AGGAGAAATGCCTGACGAAGCTCTCCA-TAMRA)-3 5 -GCTCGCCGGCTTCGA-3 5 -GGTAGGTCTGAAAGGCGAACAG-3
AJ250838
5 -(FAM)-CCACATTCTTGCAGTGAGGTCCGCT-(TAMRA)-3 5 -CCAGTGCATAGAGACTCATTCCAAA-3 5 -TGCCCATCTTTCAGAGTAGCTATGAACT-3
AF277660
5 -(FAM)-CTTTACCAGCGCGTCCTACCTTGCGACA-(TAMRA)-3 5 -GCCCTCCTCCTGGTTTCAG-3 5 -TGGCACCGCCAGCTCATT-3
AJ009800
a
Genomic DNA sequence. Forward. c Reverse. d 5-Carboxyfluorescein.
b
2.4. Oxidative burst assay Production of an oxidative burst by phorbol myristate acetate (PMA)-stimulated chicken heterophils and monocytes was measured by oxidation of 2 ,7 dichlorofluorescin-diacetate (DCFH-DA) to fluorescent DCF as described previously (He et al., 2003) with modification. One milliliter of chicken heterophils (8 × 106 cells/ml) was added to 2-ml microcentrifuge tubes and then incubated with PMA (1.62 M) and DCFH-DA (10 g/ml in final concentration) for 1 h at 41 ◦ C. The aliquots of cell cultures (150 l) were then dispensed to a black 96-well plate and the fluorescence was measured using a GENios Plus Fluorescence Microplate Reader (TECAN US Inc., Research Triangle Park, NC) at 485 nm excitation and 530 nm emission wavelengths. The relative fluorescent units (RFU) were recorded after 60 min. At least three replicates were conducted for each assay with the heterophils from each pool of chickens.
2.5. RNA isolation RNA was extracted from lysed heterophils using a RNeasyMini kit (Qiagen) following manufacturer’s instructions. Total RNA was stored at −80 ◦ C until used.
Cytokine and chemokines mRNA expression was quantitated using a well-described method. Primers and probes for cytokines, chemokines, and 28S RNA-specific amplification have been described (Kaiser et al., 2000; Kogut et al., 2003) but for clarity are provided (Table 1). The qRT-PCR was performed using the TaqMan fast universal PCR master mix and one-step RT-PCR master mix reagents (Applied Biosystems, Cheshire, UK). Amplification and detection of specific products were performed using the Applied Biosystems 7500 Fast Real-Time PCR System with the following cycle profile: one cycle of 48 ◦ C for 30 min, 95 ◦ C for 20 s, and 40 cycles of 95 ◦ C for 3 s and 60 ◦ C for 30 s. Quantification was based on the increased fluorescence detected by the 7500 Fast Sequence Detection System due to hydrolysis of the target-specific probes by the 50-nuclease activity of the rTth DNA polymerase during PCR amplification. To correct differences in RNA levels between samples within the experiment, the correction factor for each sample was calculated by dividing the mean Ct value for 28S rRNA-specific product for each sample, by the overall mean Ct value for the 28S rRNA-specific product from all samples. The corrected cytokine mean was calculated: Average of each replicate × cytokine slope 28S slope × 28S correction factor
2.6. Real-time quantitative polymerase chain reaction 2.7. Lipolysaccharide preparation Cytokine and chemokine mRNA levels in control cells and primed cells following BT treatment and stimulation with OpSE. Primer and probe sets for the cytokines and 28S were designed using the Primer Express software program (PE Applied Biosystems, Foster City, CA).
Ultra-pure lipolysaccharide (from Salmonella minnesota; LPS) was purchased from InVivoGen (San Diego, CA) and prepared in sterile physiological water as per manufacturer’s instructions.
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Table 2 Effect of aqueous BT on generation of oxidative burst by heterophils and monocytes.a BT concentration (ppm)
Heterophils
Monocytes
Relative fluorescent units (RFU)
0 4 12
Direct stimulationb (−PMA)
Primingb (+PMA)
Direct stimulationb (−PMA)
3592 ± 59 3393 ± 92 3557 ± 23
9785 ± 120 21,721 ± 244* 24,667 ± 381*
4180 ± 268 4146 ± 279 4274 ± 231
Primingb (+PMA) 17,632 ± 874 33,630 ± 210* 34,786 ± 156*
Within rows, numbers with * are significantly different (p ≤ 0.01) from the medium control cells. a Data are presented as mean ± SEM. b Direct stimulation, treatment alone stimulates cell function without an inflammatory stimulus, (−PMA); priming, treatment enhances cells responsiveness to an appropriate inflammatory stimulus (+PMA).
LPS was used at the optimal concentrations (LPS: 20 g/ml) that were previously described (Kogut et al., 2005). 2.8. MAPK family (p38 and ERK1/2) immunoassays Phosphorylation of p38 and ERK1/2 protein levels was quantitated using commercially available immunoassay kits (BioSource International, Inc., Camarillo, CA). Briefly, heterophils or monocytes (1 × 107 ) were treated with RPMI (control) or SE for 1 h at 39 ◦ C on a rocker. Following stimulation, heterophils were centrifuged (6000 × g for 5 min at 4 ◦ C), and the supernatant discarded. Cells were washed twice with ice-cold PBS (6000 × g for 10 min at 4 ◦ C) and then lysed with the appropriate freshly prepared extraction buffer (1 ml) for 30 min on ice (vortex the sample every 10 min). Supernatants were collected and stored at −70 ◦ C until the immunoassays were performed according to the manufacturer’s suggestions. The ERK samples were boiled for 5 min prior to dilution. Briefly, 100 l of freshly prepared standards and samples (diluted 1:10) were added to the appropriate wells and incubated overnight at 4 ◦ C in the dark. The initial 2 h incubation was extended to overnight to optimize sensitivity for chicken leukocytes (Genovese et al., 2007). The following morning, the plate was washed four times with wash buffer using a plate washer. The detection antibody (100 l) was added to each well except the chromogen blank and incubated for 1 h at room temperature in the dark. The plate was washed four times with the wash buffer; the HRP antibody (100 l) was added again leaving the chromogen blank, and incubated for 30 min at room temperature in the dark. The plate was washed four times; chromogen solution (100 l) was added to all wells and incubated 30 min at room temperature in the dark. Stop Solution (100 l) was added and the plate read at 450 nm within 5 min (GENios Plus Fluorescence Microplate Reader). Results were calculated using the four parameter algorithm standard curve.
30 min on a rotary shaker in a 5% CO2 incubator. The cells were then used to perform functional assays as described above. The anti-coagulated blood from 100 chickens was pooled and the peripheral blood heterophils and monocytes were isolated from each treatment group as described above. Each oxidative burst assay was conducted four times over a two-month period with pooled cells (heterophils pooled from 100 chickens for each preparation; i.e., 400 chickens in total were used as cell donors). At least three replicates were conducted for each assay with the cells from each pool of chickens. The data from these four repeated experiments were pooled for presentation and statistical analysis. The mean and standard error of the mean were calculated for each of the treatment groups. Differences between the (a) non-stimulated, (b) non-primed and (c) the primed heterophils were determined by analysis of variance. Significant differences were further separated using Duncan’s multiple range test. The data obtained using cells stimulated with aqueous BT were compared to non-stimulated control cells (ANOVA). All statistical analyses were conducted with SigmaStat 3.10 software (Systal Software, Point Richmond, CA). The phosphorylation of ERK1/2 and p38 was analyzed using a one-sided Student’s t test. 3. Results 3.1. Oxidative burst The effects of BT on the oxidative burst generated by heterophils and monocytes isolated from day-old chicks are shown in Table 2. Treatment of either cell type with the aqueous BT did not directly stimulate an oxidative burst. However, treatment of both heterophils and monocytes with BT primed for the cells for at least a 2-fold increase (p ≤ 0.01) in PMA-induced oxidative burst as compared to the age-matched control heterophils treated with tissue culture medium only.
2.9. Experimental design of functional assays 3.2. Pro-inflammatory cytokines To measure the effect of BT on heterophil and monocyte functions, cells were incubated for 2 h at 39 ◦ C in a 5% CO2 incubator with and without BT (4 or 12 ppm) and then subsequently stimulated either with RPMI medium alone or with PMA (oxidative burst) or LPS (mRNA expression) for
BT did not directly stimulate an up- or down-regulation of mRNA expression of the pro-inflammatory cytokines, IL-1 and IL-6, in heterophils and monocytes (Table 3). However, pretreatment of both heterophils and monocytes
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Table 3 Effect of aqueous BT on pro-inflammatory cytokine mRNA expression in heterophils and monocytes.a BT concentration (ppm)
Heterophils
Monocytes
IL-1 (40 − Ct ) Direct stimulationb (−LPS) 0 4 12
15.93 ± 1.20 15.84 ± 1.22 15.79 ± 1.31
BT concentration (ppm)
Heterophils
Primingb (+LPS) 15.07 ± 1.07 17.99 ± 1.21* 20.03 ± 1.18*
Direct stimulationb (−LPS)
Primingb (+LPS)
10.12 ± 0.12 10.22 ± 0.14 10.01 ± 0.20
12.21 ± 0.34 13.37 ± 0.11* 14.42 ± 0.26*
Monocytes
IL-6 (40 − Ct ) Direct stimulationb (−LPS) 0 4 12
18.83 ± 1.01 18.23 ± 1.11 18.72 ± 0.31
Primingb (+LPS) 20.09 ± 1.18 22.02 ± 1.17* 23.15 ± 0.35*
Direct stimulationb (−LPS)
Primingb (+LPS)
10.12 ± 0.06 10.22 ± 0.09 10.37 ± 0.11
12.74 ± 0.34 14.87 ± 0.27* 16.31 ± 0.43*
Within rows, numbers with * are significantly different (p ≤ 0.05) from the non-BT-treated control cells. a Data are presented as mean (40 − Ct ) ± SEM. b Direct stimulation, treatment alone stimulates cell function without an inflammatory stimulus (−LPS); priming, treatment enhances cells responsiveness to an appropriate inflammatory stimulus (+LPS).
with BT primed either cell type for a small, but significant (p ≤ 0.05), concentration-dependent increase in mRNA transcription of IL-1 and IL-6 when stimulated with LPS. 3.3. Inflammatory chemokines BT did not directly stimulate an up- or down-regulation of mRNA expression of the inflammatory chemokines, CXCLi1 and CXCLi2, in heterophils and monocytes (Table 4). However, pretreatment of both heterophils and monocytes with BT primed both cell types for a significant increase (p ≤ 0.05) in mRNA expression of both CXCLi1 and CXCLi2 when the cells were subsequently stimulated with LPS (Table 4). 3.4. Phosphorylation of p38 and ERK1/2 MAPK kinases To determine whether BT induced activation of the MAPKs, ERK1/2 and/or p38, peripheral blood-derived heterophils or monocytes were treated with 12 ppm BT or RPMI (as a vehicle control) for 10 or 20 min. In all cases, significant increases (p ≤ 0.05) in the phosphorylation of ERK1/2 and p38 were observed in response to BT treatment (Fig. 1A–D). 4. Discussion Here, for the first time, using in vitro models for measuring leukocyte function, mRNA expression, and phosphorylation of MAPK signaling proteins, we report that a group of related cationic amphipathic peptides (BT peptides), produced by a novel Gram-positive bacterium B. texasporus (Wu et al., 2005), are multifunctional modulators of avian innate immune responses. BT cationic peptides function by activating MAPK pathways (p38 and ERK1/2) and prime heterophils and monocytes for increased cellular (oxidative burst) and molecular functions (pro-inflammatory cytokine and inflammatory chemokine mRNA expression) following stimulation with inflammatory agonists. Multiple inflammatory mediators
are involved in modulating the cellular response to an infection. Inflammatory mediators that function in this modulating role are known as priming agents. Priming has a direct effect on cell shape, integrin/selectin expression, and longevity of the phagocyte, thus having a profound effect on the chemotactic, adhesive, and survival properties of the host innate cells (Condliffe et al., 1998; Rohn et al., 1999). Characteristically, the priming agent does not induce a direct functional response (Rohn et al., 1999). The priming activity of BT peptides on chicken heterophils and monocytes was verified in the present experiments. Not only did BT prime the heterophils and monocytes for an increase in transcription of pro-inflammatory cytokines induced by inflammatory agonists, but it also up-regulated expression of inflammatory chemokines mRNA. Although BT priming modulated the expression of cytokine mRNA in the leukocytes stimulated by different inflammatory agonists, BT by itself neither directly induced cell functional activity nor gene expression of either the pro-inflammatory cytokines or inflammatory chemokines. By definition, this represents true priming of the heterophils and monocytes. The practical importance of the present findings is intriguing in view of the fact that neonatal poultry exhibit a greater susceptibility to infectious diseases during the first week of life largely because of a qualitative impairment of the avian innate host defenses (Seto, 1981; Lowry et al., 1997; Wells et al., 1998). This period of transient immunologic incompetence is characterized by a functional inefficiency of heterophils and monocytes for the first 7 days of life (Lowry et al., 1997; Wells et al., 1998; He et al., 2008). We have previously demonstrated that neonatal poultry innate immunity can be effectively modulated via preventive activation of heterophils and macrophages (Kogut et al., 1993, 1994, 1997, 1998; McGruder et al., 1993, 1995a,b; Wells et al., 1998). Preventive activation provides the benefit of increased immune efficiency without the disadvantage of the potentially harmful excessive inflammation that can lead to tissue damage (Toth et al., 1987, 1988). Preventive activation involves the introduction of an
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Table 4 Effect of aqueous BT on inflammatory chemokine mRNA expression in heterophils and monocytes.a BT concentration (ppm)
Heterophils
Monocytes
CXCLi1 (40 − Ct ) Direct stimulationb (−LPS) 0 4 12
4.29 ± 0.07 4.44 ± 0.12 4.26 ± 0.09
BT concentration (ppm)
Heterophils
Primingb (+LPS) 5.56 ± 0.03 8.80 ± 0.26* 9.75 ± 0.23*
Direct stimulationb (−LPS) 5.53 ± 0.04 5.77 ± 0.13 5.75 ± 0.19
Primingb (+LPS) 6.68 ± 0.11 9.64 ± 0.05* 11.03 ± 0.82*
Monocytes
CXCLi2 (40 − Ct ) Direct stimulationb (−LPS) 0 4 12
3.07 ± 0.53 5.86 ± 0.21 5.80 ± 0.17
Primingb (+LPS) 6.35 ± 0.10 10.64 ± 0.17* 11.65 ± 0.22*
Direct stimulationb (−LPS) 8.27 ± 0.11 8.04 ± 0.07 8.13 ± 0.16
Primingb (+LPS) 10.28 ± 0.33 12.98 ± 0.42* 14.08 ± 0.26*
Within rows, numbers with * are significantly different (p ≤ 0.01) from the non-BT-treated control cells. a Data are presented as mean (40 − Ct ) ± SEM. b Direct stimulation, treatment alone stimulates cell function without an inflammatory stimulus (−LPS); priming, treatment enhances cells responsiveness to an appropriate inflammatory stimulus (+LPS).
Fig. 1. (A–D) Stimulation of MAPK signaling in heterophils and monocytes stimulated with BT peptides. Heterophils or monocytes (1 × 107 ) were cultured with either 12 ppm BT peptides or RPMI vehicle control for 10 or 20 min as described in Section 2. The expression of total phosphorylated p38 or ERK1/2 kinases was evaluated in separate ELISA kits. Data are presented as the total phosphorylated MAPK relative to the control heterophils (A = ERK1/2 and B = p38) or monocytes (C = ERK1/2 and D = p38). Columns with ** are significantly different (p ≤ 0.01) from RPMI control cells.
immunostimulant into chicks, which will induce the migration of increased numbers of primed phagocytic cells to the site of infection by a pathogenic organism (Tellez et al., 1993; Kogut et al., 1993, 1994, 1997, 1998; McGruder et al., 1993, 1995a,b; Wells et al., 1998). Therefore, based on the results from the present studies, BT peptides appear to provide a preventive activation to innate immune cells that can initiate a cell migration to the site of infection in response to infection and the increased phagocytosis and killing of the
invading bacteria. Further experiments are ongoing to test this hypothesis with other bacterial infections including Clostridium, Campylobacter, and Enterococcus. In mammals, natural and synthetic cationic antimicrobial peptides have been shown to possess two functions: (1) some direct acting antimicrobial activity (Finlay and Hancock, 2004; Hancock and Sahl, 2006) and (2) modulation of the innate host defenses (Bowdish et al., 2005; Scott et al., 2007; Akbari et al., 2008; Nijnik et al., 2010). Cationic
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amphiphilic peptides produced by host immune cells in response to infection have been found to directly stimulate the innate immune responses in mammals (reviewed by Steinstraesser et al., 2011). As presented in the current studies, BT peptides appear to have a totally unique mechanism of action from the mammalian antimicrobial peptides since they do not directly activate innate immune cells. There are no reports in the literature that we are aware of that show a priming effect by known cationic antimicrobial peptides produced by bacteria, insects, plants, birds, mammals, or humans (Hancock and Lehrer, 1998; Zasloff, 2002; Hancock, 2001; Brown and Hancock, 2006). This priming activity makes BT peptides uniquely suitable for use in neonatal chickens because of the functional inefficiency of the innate system in birds the first 7–10 days post-hatch. Mechanistically, the present results demonstrate that cytokine gene expression in avian heterophils and monocytes can be regulated by the BT peptides to allow the cells to respond in a qualitative manner to a stimulus. For example, BT peptides may play a very specialized role in the local intestinal innate responses. The increased expression of CXCLi1 and CXCLi2 shows that heterophils and monocytes can direct the recruitment of further innate immune cells that leads to the site of infection, thus increasing the ability of the host to limit the infection. Accordingly, these results imply that both cell types are proficient in amplifying the local acute inflammatory response. Furthermore, the expression of IL-1 and IL-6 in the BT-primed heterophils and monocytes would further lead to enhance bacterial clearance. We have previously shown that an increase in IL-1 and IL-6 mRNA expression is associated with increased resistance to extraintestinal SE infections in neonatal chickens (Ferro et al., 2004). One further point needs to be addressed. The BT peptides were HPLC purified and were the only peptides present with purity greater than 98%. However, since the BT peptides were purified from bacterial culture supernatants, the question arises that some pathogen-associated molecular patterns (PAMPs) were contaminants and that some of the contaminating PAMPs might have exerted some effects on monocytes/heterophils either alone or in synergy with BT peptides. Functionally, it is doubtful that BT peptides only prime innate immunity cells while PAMPs directly activate innate immunity cells. The absence of direct activation of innate immunity cells by BT peptides actually shows that the HPLC purified BT peptides are free of PAMP contamination. Furthermore, this question also promotes a very intriguing idea that BT and PAMPs may interact at the level of toll-like receptor (TLR) signaling. BT’s priming effects actually resemble those of certain “endogenous TLR ligands” which are now recognized as PAMP-sensitizing molecules rather than previously thought TLR agonists (see review by Erridge, 2010). We are currently testing the hypothesis of BT functioning as a PAMP-sensitizer. In summary, we found that in vitro pretreatment of peripheral blood heterophils and monocytes with BT peptides induced a rapid (10 min) phosphorylation and activation of the extracellular signal-regulated kinase (ERK1/2) and p38 kinase pathways. Activation of these signaling pathways leads to the priming of both heterophils and monocytes for an increased oxidative burst
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and up-regulation in transcription of the pro-inflammatory cytokines IL-1 and IL-6 and inflammatory chemokines CXCLi1 and CXCLi2 induced by inflammatory agonists. These small cationic peptides may prove useful as immune modulatory agents in immunologically inefficient neonatal poultry. Acknowledgments All experiments were conducted according to regulations established by the USDA Animal Care Committee and overseen by Dr J.A. Byrd, attending veterinarian. Mention of commercial products is for the sole purpose of providing specific information and not a recommendation or endorsement by the USDA. References Akbari, M.R., Haghighi, H.R., Chambers, J.R., Brisbin, J., Read, L.R., Sharif, S., 2008. Expression of antimicrobial peptides in cecal tonsils of chickens treated with probiotics and infected with Salmonella enterica serovar Typhimurium. Clin. Vaccine Immunol. 15, 1689–1693. Bischoff, K.M., Pishko, E.J., Genovese, K.J., Crippen, T.L., Holtzapple, C., Stanker, L.H., Nisbet, D.J., Kogut, M.H., 2001. The chicken mim-1, P33, is a heterophil chemotactic factor present in Salmonella enteritidisimmune lymphokine. J. Food Prot. 64, 1503–1509. Bowdish, D.M.E., Davidson, D.J., Scott, M.G., Hancock, R.E., 2005. Immunomodulatory activities of small host defense peptides. Antimicrob. Agents Chemother. 49, 1727–1732. Brown, K.L., Hancock, R.E.W., 2006. Cationic host defense (antimicrobial) peptides. Curr. Opin. Immunol. 18, 24–30. Condliffe, A.M., Kitchen, E., Chilvers, E.R., 1998. Neutrophil priming: pathophysiological consequences and underlying mechanisms. Clin. Sci. 94, 461–471. Erridge, C., 2010. Endogenous ligands of TLR2 and TLR4: agonists or assistants? J. Leukoc. Biol. 87, 989–999. Ferro, P.J., Swaggerty, C.L., Kaiser, P., Pevzner, I.Y., Kogut, M.H., 2004. Heterophils isolated from chickens resistant to extraintestinal Salmonella enteritidis infection express higher levels of pro-inflammatory cytokine mRNA expression following infection than heterophils from susceptible chickens. Epidemiol. Infect. 132, 1029–1037. Finlay, B.B., Hancock, R.E.W., 2004. Can innate immunity be enhanced to treat microbial infections? Nat. Rev. 2, 497–504. Genovese, K.J., He, H., Lowry, V.K., Kogut, M.H., 2007. Comparison of MAP and tyrosine kinase signaling in heterophils from commercial and wild-type turkeys. Dev. Comp. Immunol. 31, 927–933. Hancock, R.E.W., 2001. Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect. Dis. 1, 156–164. Hancock, R.E.W., Lehrer, R., 1998. Cationic peptides: a new source of antibiotics. Trends Biotechnol. 16, 82–88. Hancock, R.E.W., Sahl, H.-G., 2006. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat. Biotechnol. 24, 1551–1557. He, H., Farnell, M.A., Kogut, M.H., 2003. Inflammatory agonist stimulation and signal pathway of oxidative burst in neonatal chicken heterophils. Comp. Biochem. Physiol. Part A 135, 177–184. He, H., Lowry, V.K., Swaggerty, C.L., Ferro, P., Kogut, M.H., 2005. In vitro activation of chicken leukocytes and in vivo protection against Salmonella enteritidis organ invasion and peritoneal S. enteritidis infection-induced mortality in neonatal chickens by immunomodulatory CpG oligodeoxynucleotide. FEMS Immunol. Med. Microbiol. 43, 81–89. He, H., Genovese, K.J., Swaggerty, C.L., Nisbet, D.J., Kogut, M.H., 2008. Differential induction of nitric oxide, degranulation, and oxidative burst activities in response to microbial agonist stimulations in monocytes and heterophils from young commercial turkeys. Vet. Immunol. Immunopathol. 123, 177–185. Jiang, Y.W., Sims, M.D., Conway, D.P., 2005. The efficacy of TAMUS 2032 in preventing a natural outbreak of colibacillosis in broiler chickens in floor pens. Poult. Sci. 8, 1857–1859. Kaiser, P., Rothwell, L., Galyov, E.E., Barrow, P.A., Burnside, J., Wigley, P., 2000. Differential cytokine expression in avian cells in response to invasion by Salmonella typhimurium, Salmonella enteritidis, and Salmonella gallinarum. Microbiology 146, 3217–3226.
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Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Research paper
BT cationic peptides: Small peptides that modulate innate immune responses of chicken heterophils and monocytes Michael H. Kogut a,∗ , Kenneth J. Genovese a , Haiqi He a , Christina L. Swaggerty a , Yi Wei Jiang b a b
Southern Plains Agricultural Research Center, USDA-ARS, College Station, TX 77845, United States Department of Cell Biology & Genetics, University of North Texas Health Sciences Center, 3500 Camp Bowie, Fort Worth, TX 76107, United States
a r t i c l e
i n f o
Article history: Received 9 June 2011 Received in revised form 10 October 2011 Accepted 31 October 2011 Keywords: Antimicrobial peptide Innate immunity Heterophils Monocytes Chickens Priming
a b s t r a c t Neonatal poultry exhibit a transient susceptibility to infectious diseases during the first week of life that stems from inefficient host defense mechanisms. Yet, the initial host immune response to pathogens is a critical determinant of disease resistance and susceptibility. With this context in mind, novel ways to stimulate or modulate the hosts’ natural immune response is emerging as an important area of interest for food animal producers including the poultry industry. Specifically, we have been investigating new modulation strategies tailored around the selective stimulation of the host’s immune system, and particularly rapid acting innate immunity, as an alternative to direct targeting of microbial pathogens. One such approach that we have been investigating is the use of a group of cationic peptides produced by a Gram-positive soil bacterium, Brevibacillus texasporus (BT peptides). We have previously shown that, provided as a feed additive, BT peptides significantly induced a concentration-dependent protection against cecal colonization and extraintestinal colonization by Salmonella enterica serovar Enteritidis (SE). This protection is not the result of direct antibacterial activity of the BT peptides on the SE since the concentrations used were below the minimum inhibitory concentration for SE. We also found that BT are not absorbed in the intestine, but still induce a significant up-regulation in the functional efficiency of peripheral blood heterophils and monocytes. The mechanisms of this immune modulation are unknown. Here, using in vitro models for measuring: (1) leukocyte oxidative burst, (2) changes in leukocyte cytokine and chemokines gene expression profiles, and (3) phosphorylation of the mitogen activated protein kinases (MAPKs) in leukocytes, we evaluated the role of BT peptides as priming mediators for heterophil and monocyte responses at the level of cell function, gene transcription/expression, and cell phosphorylation following stimulation with inflammatory agonists. BT peptides primed both heterophils and monocytes for an increased oxidative burst and up-regulation in transcription of the pro-inflammatory cytokines IL-1 and IL-6 and inflammatory chemokines CXCLi1 and CXCLi2 induced by inflammatory agonists. In addition, BT peptides induced a rapid (10 min) phosphorylation and activation of the extracellular signal-regulated kinase (ERK1/2) and p38 kinase pathways in primary chicken heterophils. Taken together, we conclude that BT peptides, acting through MAPK pathways, enhance leukocyte functional and pro-inflammatory cytokine and chemokine gene transcription activities. These small cationic peptides may prove useful as immune modulators in neonatal poultry. Published by Elsevier B.V.
∗ Corresponding author. Tel.: +1 979 260 3772; fax: +1 979 260 9332. E-mail addresses: [email protected], [email protected] (M.H. Kogut). 0165-2427/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.vetimm.2011.10.023
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1. Introduction Young poultry are most susceptible to invasive pathogens during the first week post-hatch due to a functionally inefficient innate immune response (Lowry et al., 1997; Wells et al., 1998; He et al., 2008). Consequently, researchers, producers, and veterinarians are constantly developing management strategies to protect commercial and private poultry flocks against invading pathogens including Salmonella species. Research efforts in our laboratory have focused on developing immunoprophylactic strategies (Bischoff et al., 2001; Kogut, 2002; Lowry et al., 2005; He et al., 2005; Kogut et al., 2007, 2010) and selective genetics (Swaggerty et al., 2009) that prevent or control intestinal Salmonella organ and intestinal colonization in poultry. Specifically, our research has concentrated on up-regulating the innate immune response in chickens during this immunologically inefficient first week post-hatch. Recently, a novel Gram-positive bacterium Brevibacillus texasporus (ATCC PTA-5854) was isolated that produces BT, a group of structurally related cationic peptides. BT peptides were found to be highly efficacious against a natural outbreak of colibacillosis in broiler chickens based on improved performance and reduced mortality in comparison with unmedicated birds at a level (12 ppm) that was below the minimal inhibition concentration (MIC) for Escherichia coli (Jiang et al., 2005). In vitro, BT displays efficient bactericidal activity against Grampositive bacteria (MIC of 1 ppm), but a reduced efficacy against Gram-negative bacteria (MIC > 20 ppm). Interestingly, orally delivered BT seems to be completely lacking direct antibacterial activities (12 ppm; Jiang et al., 2005). In addition, chickens given BT as a feed additive for the first four days post-hatch provided protection against both cecal colonization and extra-intestinal Salmonella infections in a concentration-dependent manner and induced the up-regulation of peripheral blood heterophils and monocyte functional activities (Kogut et al., 2007, 2010). These data were intriguing because neonatal chickens are highly susceptible to systemic dissemination of enteric Salmonella infections during the first week post-hatch due to the inefficiency of the innate antibacterial defenses (Lowry et al., 1997; Wells et al., 1998). The exact mechanisms of interaction between BT peptides and heterophils and monocytes in chickens have not been elucidated. Therefore, the objective of the present experiments was to determine whether the immune modulating activities of the BT resulted from a direct stimulation of the innate immune cells or were due to a priming effect of the peptides. 2. Materials and methods 2.1. Preparation of aqueous BT peptides B. texasporus E58 cells were grown in 1 l of LB in an air shaker at 37 ◦ C for 3 days. The culture was spun in a clinical centrifuge at 3000 × g for 15 min. The supernatant was collected, and 500 g of ammonium sulfate was added and dissolved. The sample was spun in the clinical centrifuge
at 3000 × g for 15 min. The pellet was dissolved in 200 ml of distilled water. The solution was then boiled for 15 min and then cooled on ice. The sample was filtered with a 0.2m filter (Nalgene). The filtrate was mixed with 0.2 l of chloroform at room temperature for 20 min with a stir bar. The mixture was separated into two phases by centrifugation in the clinical centrifuge at 3000 × g for 15 min. The organic phase was collected and dried in a vacuum evaporator. The dried chloroform extract was dissolved in 2 ml of sterile distilled water. The solution was fractionated on a C18 reverse-phase high-performance liquid chromatography (HPLC) column by using a gradient from 30% solution B to 55% solution B (solution B was 0.075% trifluoroacetic acid in acetonitrile, and solution A was 0.1% trifluoroacetic acid in water). The resulting fractions were dried, dissolved in sterile distilled water, and analyzed with mass spectrometry for peptide identification. Peak fractions containing BT isomers were pooled and analyzed for anti-Staphylococcus aureus activity with a Kirby–Bauer assay to quantify the BT concentration (with MIC = 0.8 g/ml) before being used in the in vitro heterophil priming assays. 2.2. Experimental animals One-day-old Cobb × Ross straight-run broiler chicks were obtained from a local commercial hatchery and were placed on new pine shavings. Birds were provided water and a balanced, unmedicated ration ad libitum. The feed ration contained or exceeded the levels of critical nutrients recommended by the National Research Council (1994). 2.3. Leukocyte isolation Chicken heterophils and peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood as described previously (Kogut et al., 1995). Briefly, peripheral blood from approximately 100 chicks was pooled, mixed with 1% methylcellulose (1:1, v/v), and centrifuged at 25 × g for 15 min. The supernatant was diluted with Ca2+ - and Mg2+ -free Hanks balanced salt solution, carefully layered onto a discontinuous Histopaque gradient (specific gravity 1.077/1.119) in 50 ml conical centrifuge tubes, and centrifuged at 250 × g for 60 min. The peripheral blood mononuclear cell (PBMC) layer at the 1.077/supernatant interface was collected, washed, and resuspended in RPMI-1640. The heterophils, located below the Histopaque 1.077/1.119 interface, were collected, washed, and resuspended in RPMI-1640. Heterophils and PBMC were counted on a hemacytometer and kept on ice until used. Heterophil and PBMC preparations obtained by this method were typically >98% pure and >95% viable. Aliquots of 200 l of PBMC (1 × 107 cells/ml) were dispensed to a 96-well round-bottom plate and incubated at room temperature for 2 h. After incubation, non-adherent cells were removed by washing twice with the culture medium (Dulbecco’s Modified Eagles Medium [DMEM]) containing 10% chicken serum, antibiotics (100 U penicillin/ml and 100 g streptomycin/ml), and 1.5 mM l-glutamine. The adherent monocytes were used for the oxidative burst assay described below.
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Table 1 Real-time quantitative RT-PCR probes and primers. RNA target 28S Probe Fb Rc IL-1 Probe F R IL-6 Probe F R CXCLi1 Probe F R CXCLi2 Probe F R
Probe/primer sequence
Accession numbera
5 -(FAM)-AGGACCGCTACGGACCTCCACCA -(TAMRA)-3 5 -GGCGAAGCCAGAGGAAACT-3 5 -GACGACCGATTGCACGTC-3
X59733
5 -(FAM)-CCACACTGCAGCTGGAGGAAGCC-(TAMRA)-3 5 -GCTCTACATGTCGTGTGTGATGAG-3 , 5 -TGTCGATGTCCCGCATGA-3
AJ245728
5 -(FAM)-AGGAGAAATGCCTGACGAAGCTCTCCA-TAMRA)-3 5 -GCTCGCCGGCTTCGA-3 5 -GGTAGGTCTGAAAGGCGAACAG-3
AJ250838
5 -(FAM)-CCACATTCTTGCAGTGAGGTCCGCT-(TAMRA)-3 5 -CCAGTGCATAGAGACTCATTCCAAA-3 5 -TGCCCATCTTTCAGAGTAGCTATGAACT-3
AF277660
5 -(FAM)-CTTTACCAGCGCGTCCTACCTTGCGACA-(TAMRA)-3 5 -GCCCTCCTCCTGGTTTCAG-3 5 -TGGCACCGCCAGCTCATT-3
AJ009800
a
Genomic DNA sequence. Forward. c Reverse. d 5-Carboxyfluorescein.
b
2.4. Oxidative burst assay Production of an oxidative burst by phorbol myristate acetate (PMA)-stimulated chicken heterophils and monocytes was measured by oxidation of 2 ,7 dichlorofluorescin-diacetate (DCFH-DA) to fluorescent DCF as described previously (He et al., 2003) with modification. One milliliter of chicken heterophils (8 × 106 cells/ml) was added to 2-ml microcentrifuge tubes and then incubated with PMA (1.62 M) and DCFH-DA (10 g/ml in final concentration) for 1 h at 41 ◦ C. The aliquots of cell cultures (150 l) were then dispensed to a black 96-well plate and the fluorescence was measured using a GENios Plus Fluorescence Microplate Reader (TECAN US Inc., Research Triangle Park, NC) at 485 nm excitation and 530 nm emission wavelengths. The relative fluorescent units (RFU) were recorded after 60 min. At least three replicates were conducted for each assay with the heterophils from each pool of chickens.
2.5. RNA isolation RNA was extracted from lysed heterophils using a RNeasyMini kit (Qiagen) following manufacturer’s instructions. Total RNA was stored at −80 ◦ C until used.
Cytokine and chemokines mRNA expression was quantitated using a well-described method. Primers and probes for cytokines, chemokines, and 28S RNA-specific amplification have been described (Kaiser et al., 2000; Kogut et al., 2003) but for clarity are provided (Table 1). The qRT-PCR was performed using the TaqMan fast universal PCR master mix and one-step RT-PCR master mix reagents (Applied Biosystems, Cheshire, UK). Amplification and detection of specific products were performed using the Applied Biosystems 7500 Fast Real-Time PCR System with the following cycle profile: one cycle of 48 ◦ C for 30 min, 95 ◦ C for 20 s, and 40 cycles of 95 ◦ C for 3 s and 60 ◦ C for 30 s. Quantification was based on the increased fluorescence detected by the 7500 Fast Sequence Detection System due to hydrolysis of the target-specific probes by the 50-nuclease activity of the rTth DNA polymerase during PCR amplification. To correct differences in RNA levels between samples within the experiment, the correction factor for each sample was calculated by dividing the mean Ct value for 28S rRNA-specific product for each sample, by the overall mean Ct value for the 28S rRNA-specific product from all samples. The corrected cytokine mean was calculated: Average of each replicate × cytokine slope 28S slope × 28S correction factor
2.6. Real-time quantitative polymerase chain reaction 2.7. Lipolysaccharide preparation Cytokine and chemokine mRNA levels in control cells and primed cells following BT treatment and stimulation with OpSE. Primer and probe sets for the cytokines and 28S were designed using the Primer Express software program (PE Applied Biosystems, Foster City, CA).
Ultra-pure lipolysaccharide (from Salmonella minnesota; LPS) was purchased from InVivoGen (San Diego, CA) and prepared in sterile physiological water as per manufacturer’s instructions.
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Table 2 Effect of aqueous BT on generation of oxidative burst by heterophils and monocytes.a BT concentration (ppm)
Heterophils
Monocytes
Relative fluorescent units (RFU)
0 4 12
Direct stimulationb (−PMA)
Primingb (+PMA)
Direct stimulationb (−PMA)
3592 ± 59 3393 ± 92 3557 ± 23
9785 ± 120 21,721 ± 244* 24,667 ± 381*
4180 ± 268 4146 ± 279 4274 ± 231
Primingb (+PMA) 17,632 ± 874 33,630 ± 210* 34,786 ± 156*
Within rows, numbers with * are significantly different (p ≤ 0.01) from the medium control cells. a Data are presented as mean ± SEM. b Direct stimulation, treatment alone stimulates cell function without an inflammatory stimulus, (−PMA); priming, treatment enhances cells responsiveness to an appropriate inflammatory stimulus (+PMA).
LPS was used at the optimal concentrations (LPS: 20 g/ml) that were previously described (Kogut et al., 2005). 2.8. MAPK family (p38 and ERK1/2) immunoassays Phosphorylation of p38 and ERK1/2 protein levels was quantitated using commercially available immunoassay kits (BioSource International, Inc., Camarillo, CA). Briefly, heterophils or monocytes (1 × 107 ) were treated with RPMI (control) or SE for 1 h at 39 ◦ C on a rocker. Following stimulation, heterophils were centrifuged (6000 × g for 5 min at 4 ◦ C), and the supernatant discarded. Cells were washed twice with ice-cold PBS (6000 × g for 10 min at 4 ◦ C) and then lysed with the appropriate freshly prepared extraction buffer (1 ml) for 30 min on ice (vortex the sample every 10 min). Supernatants were collected and stored at −70 ◦ C until the immunoassays were performed according to the manufacturer’s suggestions. The ERK samples were boiled for 5 min prior to dilution. Briefly, 100 l of freshly prepared standards and samples (diluted 1:10) were added to the appropriate wells and incubated overnight at 4 ◦ C in the dark. The initial 2 h incubation was extended to overnight to optimize sensitivity for chicken leukocytes (Genovese et al., 2007). The following morning, the plate was washed four times with wash buffer using a plate washer. The detection antibody (100 l) was added to each well except the chromogen blank and incubated for 1 h at room temperature in the dark. The plate was washed four times with the wash buffer; the HRP antibody (100 l) was added again leaving the chromogen blank, and incubated for 30 min at room temperature in the dark. The plate was washed four times; chromogen solution (100 l) was added to all wells and incubated 30 min at room temperature in the dark. Stop Solution (100 l) was added and the plate read at 450 nm within 5 min (GENios Plus Fluorescence Microplate Reader). Results were calculated using the four parameter algorithm standard curve.
30 min on a rotary shaker in a 5% CO2 incubator. The cells were then used to perform functional assays as described above. The anti-coagulated blood from 100 chickens was pooled and the peripheral blood heterophils and monocytes were isolated from each treatment group as described above. Each oxidative burst assay was conducted four times over a two-month period with pooled cells (heterophils pooled from 100 chickens for each preparation; i.e., 400 chickens in total were used as cell donors). At least three replicates were conducted for each assay with the cells from each pool of chickens. The data from these four repeated experiments were pooled for presentation and statistical analysis. The mean and standard error of the mean were calculated for each of the treatment groups. Differences between the (a) non-stimulated, (b) non-primed and (c) the primed heterophils were determined by analysis of variance. Significant differences were further separated using Duncan’s multiple range test. The data obtained using cells stimulated with aqueous BT were compared to non-stimulated control cells (ANOVA). All statistical analyses were conducted with SigmaStat 3.10 software (Systal Software, Point Richmond, CA). The phosphorylation of ERK1/2 and p38 was analyzed using a one-sided Student’s t test. 3. Results 3.1. Oxidative burst The effects of BT on the oxidative burst generated by heterophils and monocytes isolated from day-old chicks are shown in Table 2. Treatment of either cell type with the aqueous BT did not directly stimulate an oxidative burst. However, treatment of both heterophils and monocytes with BT primed for the cells for at least a 2-fold increase (p ≤ 0.01) in PMA-induced oxidative burst as compared to the age-matched control heterophils treated with tissue culture medium only.
2.9. Experimental design of functional assays 3.2. Pro-inflammatory cytokines To measure the effect of BT on heterophil and monocyte functions, cells were incubated for 2 h at 39 ◦ C in a 5% CO2 incubator with and without BT (4 or 12 ppm) and then subsequently stimulated either with RPMI medium alone or with PMA (oxidative burst) or LPS (mRNA expression) for
BT did not directly stimulate an up- or down-regulation of mRNA expression of the pro-inflammatory cytokines, IL-1 and IL-6, in heterophils and monocytes (Table 3). However, pretreatment of both heterophils and monocytes
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Table 3 Effect of aqueous BT on pro-inflammatory cytokine mRNA expression in heterophils and monocytes.a BT concentration (ppm)
Heterophils
Monocytes
IL-1 (40 − Ct ) Direct stimulationb (−LPS) 0 4 12
15.93 ± 1.20 15.84 ± 1.22 15.79 ± 1.31
BT concentration (ppm)
Heterophils
Primingb (+LPS) 15.07 ± 1.07 17.99 ± 1.21* 20.03 ± 1.18*
Direct stimulationb (−LPS)
Primingb (+LPS)
10.12 ± 0.12 10.22 ± 0.14 10.01 ± 0.20
12.21 ± 0.34 13.37 ± 0.11* 14.42 ± 0.26*
Monocytes
IL-6 (40 − Ct ) Direct stimulationb (−LPS) 0 4 12
18.83 ± 1.01 18.23 ± 1.11 18.72 ± 0.31
Primingb (+LPS) 20.09 ± 1.18 22.02 ± 1.17* 23.15 ± 0.35*
Direct stimulationb (−LPS)
Primingb (+LPS)
10.12 ± 0.06 10.22 ± 0.09 10.37 ± 0.11
12.74 ± 0.34 14.87 ± 0.27* 16.31 ± 0.43*
Within rows, numbers with * are significantly different (p ≤ 0.05) from the non-BT-treated control cells. a Data are presented as mean (40 − Ct ) ± SEM. b Direct stimulation, treatment alone stimulates cell function without an inflammatory stimulus (−LPS); priming, treatment enhances cells responsiveness to an appropriate inflammatory stimulus (+LPS).
with BT primed either cell type for a small, but significant (p ≤ 0.05), concentration-dependent increase in mRNA transcription of IL-1 and IL-6 when stimulated with LPS. 3.3. Inflammatory chemokines BT did not directly stimulate an up- or down-regulation of mRNA expression of the inflammatory chemokines, CXCLi1 and CXCLi2, in heterophils and monocytes (Table 4). However, pretreatment of both heterophils and monocytes with BT primed both cell types for a significant increase (p ≤ 0.05) in mRNA expression of both CXCLi1 and CXCLi2 when the cells were subsequently stimulated with LPS (Table 4). 3.4. Phosphorylation of p38 and ERK1/2 MAPK kinases To determine whether BT induced activation of the MAPKs, ERK1/2 and/or p38, peripheral blood-derived heterophils or monocytes were treated with 12 ppm BT or RPMI (as a vehicle control) for 10 or 20 min. In all cases, significant increases (p ≤ 0.05) in the phosphorylation of ERK1/2 and p38 were observed in response to BT treatment (Fig. 1A–D). 4. Discussion Here, for the first time, using in vitro models for measuring leukocyte function, mRNA expression, and phosphorylation of MAPK signaling proteins, we report that a group of related cationic amphipathic peptides (BT peptides), produced by a novel Gram-positive bacterium B. texasporus (Wu et al., 2005), are multifunctional modulators of avian innate immune responses. BT cationic peptides function by activating MAPK pathways (p38 and ERK1/2) and prime heterophils and monocytes for increased cellular (oxidative burst) and molecular functions (pro-inflammatory cytokine and inflammatory chemokine mRNA expression) following stimulation with inflammatory agonists. Multiple inflammatory mediators
are involved in modulating the cellular response to an infection. Inflammatory mediators that function in this modulating role are known as priming agents. Priming has a direct effect on cell shape, integrin/selectin expression, and longevity of the phagocyte, thus having a profound effect on the chemotactic, adhesive, and survival properties of the host innate cells (Condliffe et al., 1998; Rohn et al., 1999). Characteristically, the priming agent does not induce a direct functional response (Rohn et al., 1999). The priming activity of BT peptides on chicken heterophils and monocytes was verified in the present experiments. Not only did BT prime the heterophils and monocytes for an increase in transcription of pro-inflammatory cytokines induced by inflammatory agonists, but it also up-regulated expression of inflammatory chemokines mRNA. Although BT priming modulated the expression of cytokine mRNA in the leukocytes stimulated by different inflammatory agonists, BT by itself neither directly induced cell functional activity nor gene expression of either the pro-inflammatory cytokines or inflammatory chemokines. By definition, this represents true priming of the heterophils and monocytes. The practical importance of the present findings is intriguing in view of the fact that neonatal poultry exhibit a greater susceptibility to infectious diseases during the first week of life largely because of a qualitative impairment of the avian innate host defenses (Seto, 1981; Lowry et al., 1997; Wells et al., 1998). This period of transient immunologic incompetence is characterized by a functional inefficiency of heterophils and monocytes for the first 7 days of life (Lowry et al., 1997; Wells et al., 1998; He et al., 2008). We have previously demonstrated that neonatal poultry innate immunity can be effectively modulated via preventive activation of heterophils and macrophages (Kogut et al., 1993, 1994, 1997, 1998; McGruder et al., 1993, 1995a,b; Wells et al., 1998). Preventive activation provides the benefit of increased immune efficiency without the disadvantage of the potentially harmful excessive inflammation that can lead to tissue damage (Toth et al., 1987, 1988). Preventive activation involves the introduction of an
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Table 4 Effect of aqueous BT on inflammatory chemokine mRNA expression in heterophils and monocytes.a BT concentration (ppm)
Heterophils
Monocytes
CXCLi1 (40 − Ct ) Direct stimulationb (−LPS) 0 4 12
4.29 ± 0.07 4.44 ± 0.12 4.26 ± 0.09
BT concentration (ppm)
Heterophils
Primingb (+LPS) 5.56 ± 0.03 8.80 ± 0.26* 9.75 ± 0.23*
Direct stimulationb (−LPS) 5.53 ± 0.04 5.77 ± 0.13 5.75 ± 0.19
Primingb (+LPS) 6.68 ± 0.11 9.64 ± 0.05* 11.03 ± 0.82*
Monocytes
CXCLi2 (40 − Ct ) Direct stimulationb (−LPS) 0 4 12
3.07 ± 0.53 5.86 ± 0.21 5.80 ± 0.17
Primingb (+LPS) 6.35 ± 0.10 10.64 ± 0.17* 11.65 ± 0.22*
Direct stimulationb (−LPS) 8.27 ± 0.11 8.04 ± 0.07 8.13 ± 0.16
Primingb (+LPS) 10.28 ± 0.33 12.98 ± 0.42* 14.08 ± 0.26*
Within rows, numbers with * are significantly different (p ≤ 0.01) from the non-BT-treated control cells. a Data are presented as mean (40 − Ct ) ± SEM. b Direct stimulation, treatment alone stimulates cell function without an inflammatory stimulus (−LPS); priming, treatment enhances cells responsiveness to an appropriate inflammatory stimulus (+LPS).
Fig. 1. (A–D) Stimulation of MAPK signaling in heterophils and monocytes stimulated with BT peptides. Heterophils or monocytes (1 × 107 ) were cultured with either 12 ppm BT peptides or RPMI vehicle control for 10 or 20 min as described in Section 2. The expression of total phosphorylated p38 or ERK1/2 kinases was evaluated in separate ELISA kits. Data are presented as the total phosphorylated MAPK relative to the control heterophils (A = ERK1/2 and B = p38) or monocytes (C = ERK1/2 and D = p38). Columns with ** are significantly different (p ≤ 0.01) from RPMI control cells.
immunostimulant into chicks, which will induce the migration of increased numbers of primed phagocytic cells to the site of infection by a pathogenic organism (Tellez et al., 1993; Kogut et al., 1993, 1994, 1997, 1998; McGruder et al., 1993, 1995a,b; Wells et al., 1998). Therefore, based on the results from the present studies, BT peptides appear to provide a preventive activation to innate immune cells that can initiate a cell migration to the site of infection in response to infection and the increased phagocytosis and killing of the
invading bacteria. Further experiments are ongoing to test this hypothesis with other bacterial infections including Clostridium, Campylobacter, and Enterococcus. In mammals, natural and synthetic cationic antimicrobial peptides have been shown to possess two functions: (1) some direct acting antimicrobial activity (Finlay and Hancock, 2004; Hancock and Sahl, 2006) and (2) modulation of the innate host defenses (Bowdish et al., 2005; Scott et al., 2007; Akbari et al., 2008; Nijnik et al., 2010). Cationic
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amphiphilic peptides produced by host immune cells in response to infection have been found to directly stimulate the innate immune responses in mammals (reviewed by Steinstraesser et al., 2011). As presented in the current studies, BT peptides appear to have a totally unique mechanism of action from the mammalian antimicrobial peptides since they do not directly activate innate immune cells. There are no reports in the literature that we are aware of that show a priming effect by known cationic antimicrobial peptides produced by bacteria, insects, plants, birds, mammals, or humans (Hancock and Lehrer, 1998; Zasloff, 2002; Hancock, 2001; Brown and Hancock, 2006). This priming activity makes BT peptides uniquely suitable for use in neonatal chickens because of the functional inefficiency of the innate system in birds the first 7–10 days post-hatch. Mechanistically, the present results demonstrate that cytokine gene expression in avian heterophils and monocytes can be regulated by the BT peptides to allow the cells to respond in a qualitative manner to a stimulus. For example, BT peptides may play a very specialized role in the local intestinal innate responses. The increased expression of CXCLi1 and CXCLi2 shows that heterophils and monocytes can direct the recruitment of further innate immune cells that leads to the site of infection, thus increasing the ability of the host to limit the infection. Accordingly, these results imply that both cell types are proficient in amplifying the local acute inflammatory response. Furthermore, the expression of IL-1 and IL-6 in the BT-primed heterophils and monocytes would further lead to enhance bacterial clearance. We have previously shown that an increase in IL-1 and IL-6 mRNA expression is associated with increased resistance to extraintestinal SE infections in neonatal chickens (Ferro et al., 2004). One further point needs to be addressed. The BT peptides were HPLC purified and were the only peptides present with purity greater than 98%. However, since the BT peptides were purified from bacterial culture supernatants, the question arises that some pathogen-associated molecular patterns (PAMPs) were contaminants and that some of the contaminating PAMPs might have exerted some effects on monocytes/heterophils either alone or in synergy with BT peptides. Functionally, it is doubtful that BT peptides only prime innate immunity cells while PAMPs directly activate innate immunity cells. The absence of direct activation of innate immunity cells by BT peptides actually shows that the HPLC purified BT peptides are free of PAMP contamination. Furthermore, this question also promotes a very intriguing idea that BT and PAMPs may interact at the level of toll-like receptor (TLR) signaling. BT’s priming effects actually resemble those of certain “endogenous TLR ligands” which are now recognized as PAMP-sensitizing molecules rather than previously thought TLR agonists (see review by Erridge, 2010). We are currently testing the hypothesis of BT functioning as a PAMP-sensitizer. In summary, we found that in vitro pretreatment of peripheral blood heterophils and monocytes with BT peptides induced a rapid (10 min) phosphorylation and activation of the extracellular signal-regulated kinase (ERK1/2) and p38 kinase pathways. Activation of these signaling pathways leads to the priming of both heterophils and monocytes for an increased oxidative burst
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and up-regulation in transcription of the pro-inflammatory cytokines IL-1 and IL-6 and inflammatory chemokines CXCLi1 and CXCLi2 induced by inflammatory agonists. These small cationic peptides may prove useful as immune modulatory agents in immunologically inefficient neonatal poultry. Acknowledgments All experiments were conducted according to regulations established by the USDA Animal Care Committee and overseen by Dr J.A. Byrd, attending veterinarian. Mention of commercial products is for the sole purpose of providing specific information and not a recommendation or endorsement by the USDA. References Akbari, M.R., Haghighi, H.R., Chambers, J.R., Brisbin, J., Read, L.R., Sharif, S., 2008. Expression of antimicrobial peptides in cecal tonsils of chickens treated with probiotics and infected with Salmonella enterica serovar Typhimurium. Clin. Vaccine Immunol. 15, 1689–1693. Bischoff, K.M., Pishko, E.J., Genovese, K.J., Crippen, T.L., Holtzapple, C., Stanker, L.H., Nisbet, D.J., Kogut, M.H., 2001. The chicken mim-1, P33, is a heterophil chemotactic factor present in Salmonella enteritidisimmune lymphokine. J. Food Prot. 64, 1503–1509. Bowdish, D.M.E., Davidson, D.J., Scott, M.G., Hancock, R.E., 2005. Immunomodulatory activities of small host defense peptides. Antimicrob. Agents Chemother. 49, 1727–1732. Brown, K.L., Hancock, R.E.W., 2006. Cationic host defense (antimicrobial) peptides. Curr. Opin. Immunol. 18, 24–30. Condliffe, A.M., Kitchen, E., Chilvers, E.R., 1998. Neutrophil priming: pathophysiological consequences and underlying mechanisms. Clin. Sci. 94, 461–471. Erridge, C., 2010. Endogenous ligands of TLR2 and TLR4: agonists or assistants? J. Leukoc. Biol. 87, 989–999. Ferro, P.J., Swaggerty, C.L., Kaiser, P., Pevzner, I.Y., Kogut, M.H., 2004. Heterophils isolated from chickens resistant to extraintestinal Salmonella enteritidis infection express higher levels of pro-inflammatory cytokine mRNA expression following infection than heterophils from susceptible chickens. Epidemiol. Infect. 132, 1029–1037. Finlay, B.B., Hancock, R.E.W., 2004. Can innate immunity be enhanced to treat microbial infections? Nat. Rev. 2, 497–504. Genovese, K.J., He, H., Lowry, V.K., Kogut, M.H., 2007. Comparison of MAP and tyrosine kinase signaling in heterophils from commercial and wild-type turkeys. Dev. Comp. Immunol. 31, 927–933. Hancock, R.E.W., 2001. Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect. Dis. 1, 156–164. Hancock, R.E.W., Lehrer, R., 1998. Cationic peptides: a new source of antibiotics. Trends Biotechnol. 16, 82–88. Hancock, R.E.W., Sahl, H.-G., 2006. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat. Biotechnol. 24, 1551–1557. He, H., Farnell, M.A., Kogut, M.H., 2003. Inflammatory agonist stimulation and signal pathway of oxidative burst in neonatal chicken heterophils. Comp. Biochem. Physiol. Part A 135, 177–184. He, H., Lowry, V.K., Swaggerty, C.L., Ferro, P., Kogut, M.H., 2005. In vitro activation of chicken leukocytes and in vivo protection against Salmonella enteritidis organ invasion and peritoneal S. enteritidis infection-induced mortality in neonatal chickens by immunomodulatory CpG oligodeoxynucleotide. FEMS Immunol. Med. Microbiol. 43, 81–89. He, H., Genovese, K.J., Swaggerty, C.L., Nisbet, D.J., Kogut, M.H., 2008. Differential induction of nitric oxide, degranulation, and oxidative burst activities in response to microbial agonist stimulations in monocytes and heterophils from young commercial turkeys. Vet. Immunol. Immunopathol. 123, 177–185. Jiang, Y.W., Sims, M.D., Conway, D.P., 2005. The efficacy of TAMUS 2032 in preventing a natural outbreak of colibacillosis in broiler chickens in floor pens. Poult. Sci. 8, 1857–1859. Kaiser, P., Rothwell, L., Galyov, E.E., Barrow, P.A., Burnside, J., Wigley, P., 2000. Differential cytokine expression in avian cells in response to invasion by Salmonella typhimurium, Salmonella enteritidis, and Salmonella gallinarum. Microbiology 146, 3217–3226.
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