Peptides 59 (2014) 94–102

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Bacterial toxin-inducible gene expression of cathelicidin-B1 in the chicken bursal lymphoma-derived cell line DT40: Functional characterization of cathelicidin-B1 Asuna Takeda a , Takashi Tsubaki a , Nozomi Sagae a , Yumiko Onda a , Yuri Inada a , Takuya Mochizuki a , Kazuo Okumura a , Sakae Kikuyama a,b , Tetsuya Kobayashi c , Shawichi Iwamuro a,∗ a

Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan Department of Biology, Faculty of Education and Integrated Arts and Sciences, Center for Advanced Biomedical Sciences, Waseda University, 2-2 Wakamatsu-cho, Shinjyuku-ku, Tokyo 162-8480, Japan c Department of Regulatory Biology, Faculty of Sciences, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan b

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Article history: Received 7 April 2014 Received in revised form 20 June 2014 Accepted 20 June 2014 Available online 28 June 2014 Keywords: Bursa of Fabricius Cathelicidin Chemotaxis DT40 cells Host defense peptides Membrane destruction

a b s t r a c t Chicken cathelicidin-B1 (chCATH-B1) is a major host defense peptide of the chicken bursa of Fabricius (BF). To investigate the mechanisms of chCATH-B1 gene expression in the BF, we focused on the DT40 cell line derived from chicken bursal lymphoma as a model for analysis. A cDNA encoding chCATH-B1 precursor was cloned from DT40 cells. The nucleotide sequence of the cDNA was identical with that of the BF chCATH-B1. A broth dilution analysis showed that the synthetic chCATH-B1 exhibited a significant defensive activity against both Escherichia coli and Staphylococcus aureus. A scanning microscopic analysis demonstrated that chCATH-B1 inhibited bacterial growth through membrane destruction with formation of blebs and spheroplasts. Limulus amoebocyte lysate assay and electromobility shift assay results revealed that chCATH-B1 bound to lipopolysaccharide (LPS) and lipoteichoic acid (LTA), which are the surface substances of the E. coli and S. aureus cell, respectively. A chemotactic assay results revealed that chCATH-B1 showed mouse-derived P-815 mastocytoma migrating activity dose-dependently but with a higher concentration, resulting in a loss of the activity. A semi-quantitative real-time RT-PCR analysis revealed that LPS stimulated chCATH-B1 gene expression in a dose-dependent manner and that the LPS-inducible chCATH-B1 gene expression was inhibited by the administration of dexamethasone. The chCATH-B1 mRNA levels in DT40 cells were also increased by the administration of bacterial LTA. The results indicate that bacterial toxins induce chCATH-B1 gene expression in the chicken BF and the peptide expressed in the organ would act against pathogenic microorganisms not only directly but also indirectly by attracting mast cells. © 2014 Elsevier Inc. All rights reserved.

Introduction Antimicrobial peptides (AMPs) are an evolutionally wellconserved component of the host innate immune system. They are present in various organisms ranging from bacteria to mammals and provide protection against environmental pathogenic microorganisms. AMPs are generally small (95% purity. Antimicrobial activities of the synthetic peptide (0–64 ␮g/ml) were monitored by incubation in the Mueller–Hinton broth (100 ␮l) with inocula (10 ␮l of 5 × 105 colony forming units/ml) from log-phase cultures of either Escherichia coli JCM5491 or Staphylococcus aureus JCM2874 strains (RIKEN BioResource Center) in 1% bovine serum albumin (BSA)-coated 96-well microtiter cell culture plates (Becton Dickinson, Franklin Lakes, NJ) for 16 h at 37 ◦ C in air. At the end of the incubation, absorbance at 595 nm (A595 ) of each well was determined using a microtiter plate reader (Bio-Rad, Hercules, CA). Scanning electron microscopy (SEM)

Materials and methods Total RNA extraction from DT40 cells The DT40 cell line (RCB1464) was purchased from RIKEN BioResource Center (Tsukuba, Japan). These cells were seeded in culture flasks (Techno Plastic Products, Trasadingen, Switzerland) containing RPMI 1640 culture medium (pH 7.4) (Nissui, Tokyo, Japan) supplemented with 4 mM l-glutamine, 10% fetal bovine serum (FBS) (SAFC Bioscience, Lenexa, KS), 1% normal chicken serum, 50 ␮M ␤-mercaptoethanol, and antibiotics (50 U/ml penicillin and 50 ␮g/ml streptomycin) (Life Technologies, Carlsbad, CA) under 5% CO2 –95% air at 37 ◦ C. For subculture, a portion of the cultures was moved to new flasks containing four volumes of fresh medium and kept under the same conditions. Total RNA was extracted from approximately 1.2 × 108 DT40 cells using the acid phenol/guanidinium/isothiocyanate procedure [14]. The total RNA samples were treated with DNase I (Life Technologies) at 37 ◦ C for 30 min, followed by phenol/chloroform extraction, and then precipitated with ethanol. The samples were dissolved in H2 O and stored at −80 ◦ C until use.

E. coli and S. aureus cells were grown in 1.5-ml tubes with 500 ␮l of tryptic soy broth (Becton Dickinson) to A600 = 0.6 and then incubated with 64 ␮g/ml chCATH-B1 for 2 and 4 h at 37 ◦ C. After incubation, the cells were harvested by gentle centrifugation, prefixed with 2.5% gluteraldehyde for 1 h, fixed in 1% osmium tetroxide for 1 h, and washed with phosphate buffer (PB). The cells were dehydrated in an ethanol series (50%, 70%, 90%, 95%, and 100%) on a nano-percolator filter (JEOL, Tokyo), followed by incubation with ethanol/t-butyl alcohol (1:1) for 1 h and three rounds of incubation with 100% t-butyl alcohol for 20 min. The samples were frozen at 4 ◦ C, lyophilized, coated with gold (15 nm) using the Quick Coater SC701 (Sanyu Electron, Tokyo), and then subjected to morphological observation using the JSM-6390LV SEM (JEOL). Bacterial LPS and LTA binding assays Binding affinity of chCATH-B1 for bacterial LPS was investigated by the chromogenic Limulus amoebocyte lysate (LAL) assay using QCL-1000 kit (Lonza, Walkersville, MD) according to the manufacturer’s instructions. The assay followed the protocol described in previous studies [35,38,46] with a slight modification. In brief, 25 ␮l of chCATH-B1 (0–100 ␮g/ml) was added in duplicate to 25 ␮l of

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0.5 endotoxin units (EU)/ml of E. coli O111:B4 LPS in a flat-bottom 96-well tissue culture plate (VTC-P96, AS ONE, Osaka, Japan). The mixture was then incubated for 10 min at 37 ◦ C, followed by incubation with 50 ␮l of the LAL reagent in the kit for exactly 10 min. Then, 100 ␮l of the chromogenic substrate (Ac-Ile-Glu-Ala-Arg-pnitroanilide) was added and incubation was continued. Exactly 8 min later, the reaction was stopped by addition of 100 ␮l of the stop reagent (10% w/v SDS). At the end of incubation, A410 of each well was monitored. Binding affinity of chCATH-B1 for bacterial lipoteichoic acid (LTA) was determined by forming chCATH-B1–LTA complexes and observing shifts in their electrophoretic mobility during native polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions [26]. Mixtures containing increasing amounts of S. aureus LTA (0–4 ␮g) (Sigma–Aldrich, St. Louis, MO) and chCATH-B1 (10 ␮g) in 50 mM PB were incubated at 37 ◦ C for 15 min on a 20 ␮l scale, and subsequently separated by native PAGE using SuperSep 7.5% gels (Wako, Osaka). 0.1 M Tris–HCl (pH 7.8) and 0.068 M glycine–0.053 M Tris–HCl (pH 8.9) were used as the anode and cathode running buffers, respectively. The gels were stained using the Silver Stain II kit (Wako). During native PAGE, proteins/peptides with higher isoelectric points migrate toward the cathode, and therefore cannot enter the gel. Thus, unbound chCATH-B1 could not enter the gels under the experimental conditions. When chCATHB1 bound to LTA, the chCATH-B1–LTA complexes were detected by observing their appearance in the gel with increasing anionic mobility. Chemotactic assay and cytotoxic assay In this study, the mouse-derived mastocytoma cell line P-815 (RIKEN BioResource Center) was used for mast cell migration assay. We have previously shown that this cell line could be used for substitution of mast cells [18]. Different concentrations of chCATH-B1 (500 ␮l) were diluted in RPMI 1640 medium containing 10% FBS and the antibiotics and applied into each well of 24-well culture plates (Corning Inc., Corning, NY). Following this, a 24-well chemotaxicell with an 8-␮m pore-size polycarbonate membrane (Kurabo, Osaka) was placed into each well and 500 ␮l of P-815 cell suspension (2.5 × 104 cells) was added into each insert. After incubation for 3 h at 37 ◦ C in an atmosphere of 5% CO2 , non-adherent cells were aspirated from the insert, and cells adherent to the upper surface of the membrane were removed by scraping with a cotton bud. Migrated cells on the lower surface of the membrane were fixed with methanol for 5 min and stained with 0.05% toluidine blue (Wako) for 10 min. The membranes were mounted on glass slides using routine histological methods, and the total number of mast cells that migrated across the membrane was counted under a light microscope. To assess cytotoxic effects of chCATH-B1 on mastocytoma, a standard MTT assay was performed according to our previous study [18] with a slight modification. Briefly, as a reference, 3 × 104 P-815 or COS-7 (RIKEN Bioresource Center) cells per well were cultured on a collagen-coated 96-well microtiter cell culture plate (Thermo Fisher Scientific, Waltham, MA) containing 100 ␮l of RPMI 1640 medium or MEM (Invitrogen, Carlsbad, CA), respectively, supplemented with 10% FBS and the antibiotics at 37 ◦ C overnight in an atmosphere of 5% CO2 . Then, the medium was replaced to a fresh one containing chCATH-B1 at the final concentration ranging from 0 to 40 ␮g/ml, lysozyme (Sigma–Aldrich) ranging from 0 to 120 ␮g/ml (negative control), or 0.1% Triton X-100 (positive control) and was incubated for 3 h and 6 h. Aliquots (10 ␮l) of 0.5% (w/v) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Wako, Osaka, Japan) were added to each well and incubated for 4 h; then aliquots (100 ␮l) of a lysis buffer, 6 N HCl/isopropanol (0.34/99.66, v/v), were added and incubated for

another 15 h. Finally, A570 of the specimens was measured using a microtiter plate reader. Semi-quantification of chCATH-B1 mRNA expression levels using real-time RT-PCR DT40 cell cultures (20 ml; approximately 6.6 × 106 cells) in 175cm2 flasks were immersed in the following: (1) 100 ng/ml of E. coli O11 LPS (Sigma–Aldrich) for 0, 3, 6, 12, and 24 h; (2) 0, 1, 10, 100, and 1000 ng/ml LPS for 6 h; (3) a combination of 100 ng/ml LPS and 10−7 or 10−6 M dexamethasone (Dex; Wako) for 6 h, and (4) 100 ng/ml of S. aureus LTA (Sigma–Aldrich) for 0, 3, 6, 12, and 24 h. All cultures were performed under the same conditions described in “Total RNA extraction from DT40 cells” section. At the end of incubation, the cells were harvested in a 50-ml conical tube by gentle centrifugation and rinsed with PBS. Total RNA was extracted from the cells using the FastPure RNA kit (Takara, Ohtsu, Japan) and diluted to 100 ng/␮l with RNase-free H2 O. Total RNA (300 ng) from the bacterial toxin-treated DT40 cell samples was reverse transcribed to single strand cDNA using the PrimeScript RT reagent Kit (Takara) and 5 ␮M random hexamer and 2.5 ␮M oligo-dT primers in 10 ␮l reaction volume under the following conditions: 37 ◦ C for 15 min for reverse-transcription and 85 ◦ C for 85 s to inactive the reverse transcriptase. The cDNA samples were kept at 4 ◦ C until used. For semi-quantitative real-time RT-PCR, 2 ␮l of the 1st strand cDNA sample was mixed with the THUNDERBIRD Probe qPCR Mix (Toyobo, Osaka), specific forward (5 -GCATCTGGGAGTGGTTGAATG-3 ) and reverse (5 -AGGCAGAAGGGACGTTTATT-3 ) primers (300 nM; Life Technologies), gene-specific TaqMan probe (5 -ATCAGGAAGCGCCTGCGGCAG-3 ) (200 nM; Life Technologies), and ROX reference dye II in the kit. A Prism 7500 real-time PCR system (Life Technologies) was used to amplify the chCATH-B1 gene from each DT40 cell sample in triplicate on a 96-well reaction plate using the following protocol: 1 min denaturation at 95 ◦ C, then 50 cycles of 15 s denaturation at 95 ◦ C and 40 s annealing and extension at 50 ◦ C. A 18S ribosomal RNA gene was amplified using the Pre-Developed TaqMan Assay Reagent (Life Technologies) instead of the primers and TaqMan probe in every sample as an endogenous control for data normalization. Relative quantification studies were performed with the collected data using the Prism 7500 System SDS software 1.3 (Life Technologies). Statistical analyses Statistical analyses for the antimicrobial, chemotactic, and cytotoxic assays and chCATH-B1 mRNA semi-quantification were performed employing the analysis of variance (ANOVA), followed by a multiple comparison test using the Tukey–Kramer method. A value of P < 0.05 was considered statistically significant.

Results Molecular cloning of chCATH-B1 cDNA By RT-PCR using a set of chCATH-B1-specific primers, multiplesized cDNAs were amplified from DT40 cell total RNA (Fig. 1A). An appropriate-sized (850 bp) band was purified from agarose gel and subjected to nucleotide sequence analysis. It revealed that the clone consisted of 815 bp and contained a 5 -untranslated region (UTR) of 2 bp, an ORF of 780 bp including a stop codon, and a 3 -UTR of 33 bp. The nucleotide and deduced amino acid sequences exhibited 100% identities with those of the G. gallus BF chCATH-B1 in the GenBank database (Acc. No. AB307733). The deduced amino acid sequence and putative structural domains are indicated in

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Fig. 1. Expression of chicken cathelicidin-B1 (chCATH-B1) precursor mRNA in the chicken bursal cell line DT40 (A) and the predicted amino acid sequence and structural domains encoded by the mRNA (B). An aliquot of total RNA from DT40 cells was subjected to RT-PCR with a set of primers specific for the amplification of chCATH-B1. The products were separated on an agarose gel and an appropriate sized band (indicated by arrow) was subjected to nucleotide sequence analysis. The nucleotide sequence has been deposited in the GenBank database with the accession number AB915170. MM, molecular marker.

Fig. 1B. The nucleotide sequence of the DT40 cell chCATH-B1 has been deposited to the GenBank database (Acc. No. AB915170). Antimicrobial activities A synthetic replicate of chCATH-B1 demonstrated antimicrobial activities against both Gram-negative and -positive bacteria (Fig. 2). The synthetic peptide was significantly active against E. coli

at lower concentrations compared with that against S. aureus. However, the minimal inhibitory concentrations (MICs) of chCATH-B1 against E. coli and S. aureus could be considered almost similar to each other, and the value (64 ␮g/ml) was calculated to be 12.8 ␮M, which was 5–10-fold higher than that obtained by Goitsuka et al. [9], i.e., 2.5 ␮M for E. coli and 1.25 ␮M for S. aureus. These variations may have been caused by the differences in assay conditions, such as the concentrations of bacterial inocula and incubation periods, between the studies. SEM analysis Morphological observation of bacterial cells is a persuasive method for the study of mode of action of AMPs [13,20]. We used SEM to observe alterations in the morphology of E. coli and S. aureus cells after exposure to chCATH-B1. A progression of changes occurred on the surfaces of E. coli cells treated with the antimicrobial peptide compared with the control cells (Fig. 3A). These changes were characterized by the appearance of discrete blebs on the cell surface when treated with chCATH-B1 for 2 h (Fig. 3B1 and B2), followed by a marked deformation such as the cell enlargement or cell fragmentation with the formation of multiple blebs and spheroplasts after two more hours (Fig. 3C1 and C2). Likewise, chCATH-B1 treatment resulted in time-dependent morphological changes in S. aureus cells (Fig. 3D–F). Incubation with chCATH-B1 for 2 h resulted in the formation of small blebs on the cell surface and enlargement of the cells (Fig. 3E1 and E2). Subsequently, further enlargement of the cells with spheroplast formation was also observed (Fig. 3F1 and F2). Binding of chCATH to bacterial LPS and LTA

Fig. 2. Effect of various concentrations of synthetic chCATH-B1 peptide on the growth of Gram-negative bacterium E. coli strain JCM5491 (A) and Gram-positive bacterium S. aureus strain JCM2784 (B). Cells of each bacterial strain were incubated with chCATH-B1 for 16 h at 37 ◦ C. Points and vertical lines represent the means and standard error of the mean (SE), respectively (n = 3–4). In each panel, the values with the same superscript are not significantly different at the 5% level.

Interaction with bacterial cell wall molecules such as LPS and LTA, the distinct components of Gram-negative and Gram-positive bacteria, respectively, may be a trigger for the modes of antimicrobial action of typical AMPs. To assess the binding affinity of chCATH-B1 for LPS, the chromogenic LAL assay was performed. This assay is a standard method for the detection and quantification of LPS [27]. chCATH-B1 showed a dose-dependent binding affinity for LPS, and 50 ␮g/ml of the peptide suppressed LPS-derived chromogenesis almost completely, suggesting that most of the LPS molecules within the reaction (0.25 EU/ml ≈ 25 pg/ml) were bound to chCATH-B1 (Fig. 4A). A similar result was observed under different concentration conditions (0.05 EU/ml and 0.5 EU/ml) of LPS (data not shown). To assess the affinity of chCATH-B1 binding to LTA, electrophoretic mobility shift assay was performed. In the assay, chCATH-B1 was incubated with bacterial LTA and

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Fig. 3. Morphological abnormalities of CATH-B1-treated bacterial cells. Aliquots of the E. coli strain JCM5491 (A–C) or S. aureus strain JCM2784 (D–F) at the mid-logarithmic growth phase were incubated at 37 ◦ C with chCATH-B1 at a concentration of 64 ␮g/ml for 0 h (A and D), 2 h (B1, B2, E1, and E2), and 4 h (C1, C2, F1, and F2) and then subjected to SEM analysis. Abnormally shaped cells with blebs (indicated by arrows) and spheroplasts are seen in each panel except (A) and (D). Bars, 1 ␮m.

then the chCATH-B1-LTA complexes were resolved using a native PAGE. LTA caused increased anionic mobility of chCATH-B1 during electrophoresis under non-dissociating conditions and the chCATH-B1-derived bands on the gels became stronger with the concentration of LTA (Fig. 4B). These results provided the direct evidence of the binding ability of chCATH-B1 to bacterial LPS and LTA.

the optimal concentrations of 5–10 ␮g/ml. Higher concentration of chCATH-B1 suppressed migration, indicating that a threshold concentration of the peptide is needed to trigger mast cell migration. These features in the peptide concentrations showed good agreement with those in the results from a previous similar assay of LL37 [28]. No significant cytotoxic effect of chCATH-B1 on either P-815 (Fig. 5B) or COS-7 (data not shown) cells was observed under the assay conditions.

Chemotactic assay and cytotoxic assay The ability of chCATH-B1 to induce the migration of mast cells was evaluated. As shown in Fig. 5A, when different concentrations of chCATH-B1 were added to the lower compartment, P-815 mastocytoma cells migrated toward this peptide. The dose dependence of mast cell migration toward chCATH-B1 was observed with

LPS-inducible chCATH-B1 gene expression and its suppression by Dex Total RNA specimens isolated from DT40 cells cultured in medium containing 100 ng/ml LPS for 3, 6, 12, or 24 h were subjected to semi-quantitative real-time RT-PCR. As shown in Fig. 6,

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Fig. 4. The binding of chCATH-B1 to LPS (A) and LTA (B), a Gram-negative and -positive bacterial cell wall components, respectively. The binding affinity of chCATH-B1 for LPS was monitored by the chromogenic Limulus amoebocyte lysate assay. Mixtures of LPS (0.25 EU/ml) and chCATH-B1 (0–50 ␮g/ml) were incubated for 10 min at 37 ◦ C, followed by incubation with the amoebocyte lysate reagent for 10 min. Then, the chromogenic substrate was added and incubated for 8 min. The reaction was stopped by addition of the stop reagent, and A410 of each sample was monitored. (−) indicates the absence of LPS in the reaction. The LTA-binding activity of chCATH-B1 was evaluated using mobility shift assays under non-denaturating conditions. Mixtures of S. aureus LTA (0–4 ␮g) and chCATH-B1 (10 ␮g) were incubated at 37 ◦ C for 15 min, and the chCATH-B1 and LTA complexes were subjected to native PAGE followed by silver staining.

Fig. 5. Effects of chCATH-B1 on cell migration (A) and cell survival (B) of mast cells. In (A), cells of mastocytoma P-815 (2.5 × 104 cells/500 ␮l) placed in the culture inserts (upper compartment) of 24-well culture plates were allowed to migrate toward 2.5–20 ␮g/ml of CATH-B1 or medium alone in each well (lower compartment) for 3 h at 37 ◦ C. Mast cell migration was assessed by counting the number of cells that passed through the polycarbonate membrane. Points and vertical lines represent the means and SE, respectively (n = 3). The values with the same superscript are not significantly different at the 5% level. In (B), P-815 cells (3 × 104 cells/well) were cultured on a collagen-coated 96-well culture plate with 0–40 ␮g/ml of chCATH-B1 for 3 h (white columns) or 6 h (black columns) at 37 ◦ C. Then, the cells were subjected to the standard MTT assay. Survival rates were expressed by the MTT reduction values that were calculated from the relative levels obtained from the control (0 dose for 3 h incubation) cells. Columns and vertical bars represent the means and SE, respectively (n = 4). No significant cytotoxic effect was detected in chCATH-B1. PC, positive control (0.1% Triton X-100, 6 h).

Fig. 6. Effects of LPS and Dex on the steady-state levels of chCATHB1 mRNA in DT40 cells. The DT40 cells incubated with 100 ng/ml of E. coli O11 LPS for various time (A), or in various concentrations of LPS for 6 h (B). The cells were incubated for 6 h with LPS (100 ng/ml) alone, Dex (10−7 or 10−6 M) alone, or a combination of LPS and Dex (C). After incubations, total RNA was extracted from the cells and subjected to semi-quantitative real-time RT-PCR. The amounts of chCATH-B1 mRNA were normalized with respect to the amount of 18S ribosomal RNA in each sample. These values were calculated from the relative levels to those of the control (0 ng/ml LPS for 0 h). Columns and vertical lines represent the means and SE, respectively, of triplicate determination averaged from two separate determinations. The values with the same superscript are not significantly different at the 5% level. L, 100 ng/ml LPS; D, 10−7 M Dex; 10D, 10−6 M Dex.

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Fig. 7. Time-course of the effects of LTA on the steady-state levels of chCATHB1 mRNA in DT40 cells. Cells were immersed in 100 ng/ml of S. aureus LTA for 0, 3, 6, 12, and 24 h. Other details are as described in Fig. 6. Columns and vertical lines represent the means and SE, respectively, of triplicate determination averaged from two separate determinations. The values with the same superscript are not significantly different at the 5% level.

their chCATH-B1 mRNA levels were increased to 16.5-fold after 3 h and then gradually decreased to 14.3-, 11.7-, and 9.0-fold after 6, 12 and 24 h compared with the control (0 h) level, respectively (Fig. 6A). In the dose-dependent experiments, DT40 cells were immersed in 1, 10, 100, and 1000 ng/ml of LPS for 6 h and their chCATH-B1 mRNA levels were increased 0.9-, 2.5-, 10.8-, and 22.0fold compared with the control (0 ng/ml), respectively (Fig. 6B). Although chCATH-B1 mRNA expression was not affected by the Dex treatments, LPS-inducible chCATH-B1 mRNA expression was suppressed by 10−7 and 10−6 M Dex to 0.52- and 0.36-fold of the control (LPS alone) level, respectively (Fig. 6C). Effects of LTA on chCATH-B1 mRNA expression To evaluate the effects of Gram-positive bacteria-derived LTA on the expression of chCATH-B1 mRNA in DT40 cells, this pilot experiment was performed. Treatment with LTA (100 ng/ml) for 3 h induced 4.0-fold increase in chCATH-B1 mRNA levels with a subsequent decrease to basal expression levels at 6 h. However, the mRNA levels increased once again at 12 and 24 h to 2.0- and 2.5fold as compared with the control, respectively, but the difference was not significant (Fig. 7). Discussion Cathelicidins are a major family of HDPs in vertebrate animals. The peptides belonging to this family are synthesized as prepropeptides. These prepro-sequences are highly conserved among species, except for the carboxyl terminal sequence of the prepropeptides, from which the biologically active mature peptides are generated. In chicken, four kinds of cathelicidin family peptides, fowlicidins-1–3 and chCATH-B1, have been identified. These peptides have tissue-specific gene expression patterns: fowlicidins-1–3 exhibit a similar tissue expression pattern with the highest expression levels in the bone marrow and lung, whereas chCATH-B1 was synthesized most abundantly in the BF [1,9]. To study the role of HDPs in the BF, we focused on chCATH-B1 and cloned its cDNA from the bursal B lymphoma-derived cell line DT40, demonstrating that the cell line was potentially useful for primary studies of chCATH-B1 expression mechanisms.

Prior to studying the mechanisms of chCATH-B1 expression regulation, we investigated various biological functions of chCATH-B1 using a synthetic replicate of the peptide. Although the previous study describes the antimicrobial properties of chCATH-B1 [9], its mechanisms have not been determined yet. In this study, we confirmed anti-Gram-negative and -positive bacterial activities of the chCATH-B1. Subsequent SEM analyses revealed that chCATHB1 treatment brought about marked morphological abnormalities such as the spheroplast formation, and the cell enlargement and deformation. A spheroplast is a bacterial, yeast, or fungal cell of which rigid cell wall was partially removed to form a membranebound cell with a spherical shape. Therefore, chCATH-B1 was speculated to attack the cell wall of either E. coli or S. aureus at least as a part of the mode of antimicrobial action. Similar morphological abnormalities, including spheroplast formation, were observed in the cells treated with other antibiotics [16,17]. The results obtained from the SEM analyses demonstrated the presence of a substance(s) on the surfaces of E. coli and S. aureus cells that possesses binding affinity to chCATH-B1. It is known that cathelicidins and cathelicidin-related peptides have abilities to bind and/or neutralize bacterial toxins such as LPS and LTA from the cell walls of Gram-negative and -positive bacteria, respectively [30,35,38,41,46]. However, although there is direct evidence for the binding of cathelicidins to LPS, little is known about LTA-binding ability of the peptides. In this study, we showed evidence of the binding of chCATH-B1 not only to LPS but also to LTA. Based on the findings, we hypothesize that chCATH-B1 was attracted by the bacterial cell wall substances, then bound to them, broke the cell walls, and caused formation of spheroplasts. Other functions of cathelicidins including chemotaxis of neutrophils, eosinophils, T-cells, and mast cells, have been reported [8,28,44]. In addition, a recent study has shown that chicken cathelicidin/fowlicidin-1 was chemotactic to neutrophils, but not to lymphocytes and monocytes [2]. Mast cells play a critical role in immune response and inflammation [10,25] and reside in tissues throughout the body, particularly in tissues associated with structures such as blood vessels and nerves, and in tissues that interface with the external environment. Although BF can be regarded as one of such interface, few mast cells were observed in the BF of normal adult chickens [42,47]. However, the number and activity of mast cells in the BF were markedly increased after infection with very virulent infectious bursal disease virus (vvIBDV) [42]. In this study, we demonstrated the ability of chCATH-B1 to induce mast cell migration using mastocytoma cells, suggesting that chCATH-B1 may attract mast cells into the chicken BF at the time of infection by pathogenic organisms. It has been known that pathogen-associated molecular patterns (PAMPs) such as LPS induce the expression of HDP mRNAs, including cathelicidins, in various cells such as epithelial cells, endometrial cells, keratinocytes, and macrophages [15,24,30,34]. In most of the cases, expression of HDP mRNAs are speculated to be regulated by a nuclear transcription factor NF-␬B-dependent cascade that is triggered through the binding of PAMPs to pattern recognition receptors such as toll-like receptors [7]. In some cases, PAMPs-dependent HDP gene expression was inhibited by corticosteroids. These molecules are known to increase I␬B expression, which suppress the NF-␬B-dependent cascade [6,21,22]. In this study, we observed strong effects of LPS on chCATH-B1 mRNA expression and its suppression by Dex in DT40 cells. In addition, this study showed an LTA-dependent chCATH-B1 mRNA expression. These findings demonstrate that the BF may respond to PAMPs and express the chCATH-B1 gene in a similar manner to a typical HDP gene expression at the time of pathogenic inflammation. However, no obvious NF-␬B-binding motif (GGGACTTTCC) was found within the promoter region of the chCATH-B1 gene (GenBank Acc. No. AB308318). It has been known that in addition to the

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NF-␬B pathway, the mitogen-activated protein kinase (MAPK) signal transduction pathway is involved in HDP regulation [23]. Therefore, the involvement of this pathway in chCATH-B1 regulation should be investigated in the future. It is noteworthy that although B cells were easily found in normal chicken bursal follicles and bone marrow, expression of cathelicidin genes was not detected in the B-lymphocytes of those organs. In the chicken BF, chCATH-B1 gene expressed in interfollicular secretory enterocytes and mature chCATH-B1 peptide was observed to be deposited on the fibrillar network surrounding the basolateral M cell surfaces covering the bursal lymphoid follicles. Therefore, it was hypothesized that chCATH-B1 precursor protein was secreted into the bursal lumen and cleaved to the bioactive mature peptide after uptake via pinocytosis by neighboring M cells [9]. Fowlicidin-2/CATH-2 was shown to be localized in the granules of chicken heterophils, which are equivalent to mammalian neutrophils, but not in B cells in the bone marrow. It was processed into a mature form upon stimulation with bacterial LPS [40]. It has not yet been clearly demonstrated whether DT40 cells synthesize chCATH-B1 as a precursor or a mature form. Experiments are underway to answer to this question. In summary, this study reported the molecular cloning of chCATH-B1 mRNA from the cells of the chicken BF lymphomaderived cell line DT40 and its preinflammatory moleculesdependent expression and functional characterization of the chCATH-B1 peptide. It is now widely accepted that HDPs, including cathelicidins, act as a bridge between the innate and adaptive immune responses. Therefore, the BF and chCATH-B1 may be key factors to address the development of the innate and adaptive immune system in vertebrates, and the present study showed the usefulness of DT40 cells for this analysis. Author contribution AT, TT: collection and assembly of data, data analysis and interpretation; NS, YO, YI, TM, KO: data collection; SK, TK: data analysis and interpretation, financial support, and manuscript writing; SI: conception and design, financial support, assembly data, data analysis and interpretation, manuscript writing, and final approval of manuscript. Acknowledgements This work was supported by Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science to TK, SK, and SI (23570068 and 24570077) and by a grant from Faculty of Science, Toho University (21-301-04) to SI. The authors would like to thank Enago (www.enago.jp) for the English language review. References [1] Achanta M, Sunkara LT, Dai G, Bommineni YR, Jiang W, Zhang G. Tissue expression and developmental regulation of chicken cathelicidin antimicrobial peptides. J Anim Sci Biotechnol 2012;3:5. [2] Bommineni YR, Pham GH, Sunkara LT, Achanta M, Zhang G. Immune regulatory activities of fowlicidin-1, a cathelicidin host defense peptide. Mol Immunol 2014;59:55–63. [3] Buerstedde JM, Reynaud CA, Humphries EH, Olson W, Ewert DL, Weill JC. Light chain gene conversion continues at high rate in an ALV-induced cell line. EMBO J 1990;9:921–7. [4] Buerstedde JM, Takeda S. Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell 1991;67:179–88. [5] Cunliffe RN, Mahida YR. Expression and regulation of antimicrobial peptides in the gastrointestinal tract. J Leukoc Biol 2004;75:49–58. [6] Duits LA, Rademaker M, Ravensbergen B, van Sterkenburg MA, van Strijen E, Hiemstra PS, et al. Inhibition of hBD-3, but not hBD-1 and hBD-2, mRNA expression by corticosteroids. Biochem Biophys Res Commun 2001;280:522–5. [7] Froy O. Regulation of mammalian defensin expression by Toll-like receptor-dependent and independent signalling pathways. Cell Microbiol 2005;7:1387–97.

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Bacterial toxin-inducible gene expression of cathelicidin-B1 in the chicken bursal lymphoma-derived cell line DT40: functional characterization of cathelicidin-B1.

Chicken cathelicidin-B1 (chCATH-B1) is a major host defense peptide of the chicken bursa of Fabricius (BF). To investigate the mechanisms of chCATH-B1...
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