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Molecular cloning, characterization and mRNA expression of duck interleukin-17F
Veterinary Immunology and Immunopathology 164 (2015) 194–200
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Short communication
Molecular cloning, characterization and mRNA expression of duck interleukin-17F Woo H. Kim a,1 , Cherry P. Fernandez a,1 , Joyce Anne R. Diaz a , Jipseol Jeong b , Suk Kim a , Hyun S. Lillehoj c , Hong H. Chang d , Wongi Min a,∗ a b c d
College of Veterinary Medicine & Institute of Animal Medicine, Gyeongsang National University, Jinju 660-701, Republic of Korea Environmental Health Research Department, Environmental Research Complex, Incheon 404-708, Republic of Korea Animal Biosciences and Biotechnology Laboratory, BARC, ARS, USDA, Beltsville, MD 20705, USA Department of Animal Science, College of Agriculture, Gyeongsang National University, Jinju 660-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 11 November 2014 Received in revised form 7 February 2015 Accepted 19 February 2015 Keywords: Duck Cytokine Salmonella infection Interleukin-17F
a b s t r a c t Interleukin-17F (IL-17F) is a proinflammatory cytokine that plays an important role in gut homeostasis. A full-length duck IL-17F (duIL-17F) cDNA with a 510-bp coding region was identified in ConA-activated splenic lymphocytes. duIL-17F is predicted to encode 166 amino acids, including a 26-amino acid signal peptide, a single N-linked glycosylation site, and six cysteine residues that are conserved in mammalian IL-17. duIL-17F shares 77.5% amino acid sequence identity with chicken IL-17F (chIL-17F), 37–46% with corresponding mammalian homologues, and 53.5% with the previously described duck IL-17A (duIL-17A). The duIL-17F transcripts were expressed in a wide range of untreated tissues; levels were highest in the liver and moderate in the thymus, bursa, kidney, and intestinal tissues. Expression levels of duIL-17F transcript were slightly up-regulated in ConA- and LPS-activated splenic lymphocytes but not in poly I:C stimulated cells. duIL17F forms heterodimers with duIL-17A. Recombinant duIL-17F, like duIL-17A, induced IL-1, IL-6, and IL-8 expression in duck embryonic fibroblasts (DEFs). duIL-17A, but not duIL-17F expression, was significantly up-regulated in the liver and spleen of Salmonella Typhimurium-infected ducks. Further analysis of the contributions of IL-17F to different Salmonella spp. or other disease models will be required to expand our understanding of its biological functions. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The IL-17 family consists of six cytokine members (IL17A to IL-17F), which have been shown to have varying biological roles and degrees of intermolecular amino acid
∗ Corresponding author at: College of Veterinary Medicine, Gyeongsang National University, 501 Jinju-daero, Jinju, Gyeongnam 660-701, Republic of Korea. Tel.: +82 55 772 2357; fax: +82 55 772 2349. E-mail address: [email protected] (W. Min). 1 Woo H. Kim and Cherry P. Fernandez contributed equally to this work. http://dx.doi.org/10.1016/j.vetimm.2015.02.007 0165-2427/© 2015 Elsevier B.V. All rights reserved.
sequence homology in mammals. These six IL-17-related cytokines form homodimers or heterodimers and generally function through engagement of the IL-17 receptor (IL-17R) family, which includes five molecules (IL-17RA to IL-17RE) (Iwakura et al., 2011). IL-17 cytokines play important roles in host defense against variety of pathogens by inducing the generation of proinflammatory cytokines and chemokines, and have been implicated in autoimmune diseases, inflammatory diseases, and tumors (Iwakura et al., 2011; O’Sullivan et al., 2014). The best-studied members of the IL-17 family are IL-17A and IL-17F which share high amino acid sequence identity (approximately 50%)
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and bind the same receptors. Thus, these two cytokines are likely to have similar and/or overlapping biological activities (Moseley et al., 2003; Min et al., 2013). Although they have been shown to have some similar functions, IL17A and IL-17F have been suggested by some studies to be differentially regulated and to have their own distinct activities (Yang et al., 2008; Ishigame et al., 2009). IL-17F was initially identified in activated T cells and monocytes using the human IL-17A genomic sequence (Starnes et al., 2001). Unlike IL-17A, IL-17F is generally expressed in a variety of cell types and tissues, suggesting that IL-17F is involved in a broader range of biological functions (Gomez-Rodriguez et al., 2009; Kim et al., 2012). Antiangiogenic function of IL-17F has been described in endothelial cells and hepatocarcinoma cells (Starnes et al., 2001; Xie et al., 2010) and defective expression of IL-17F has been implicated in bronchial asthma (Kawaguchi et al., 2001), antigen-induced allergic response (Oda et al., 2005), chronic obstructive pulmonary disease (Hizawa et al., 2006), psoriasis (Fujishima et al., 2010), chronic intestinal inflammation (Leppkes et al., 2009), and rheumatoid arthritis (Hurst et al., 2002). The avian immune system provides an important model for the study of basic and applied immunology. Due to the general lack of cross-reactivity and the low level of sequence conservation between avian and mammalian cytokines (Staeheli et al., 2001; Starnes et al., 2001), identification of immune-related genes has been challenging. The recent dramatic growth of the duck industry is anticipated to increase the occurrence of diseases including Salmonella infection (Cha et al., 2013). Recently, duIL-17A was identified in ConA-activated splenic lymphocytes (Yoo et al., 2009) leading us to become interested in identify and characterize the duIL-17F. In this study, we identified a full-length duIL-17F cDNA. Through reverse transcription quantitative real-time RT-PCR and Western blot analysis, the tissue distribution of duIL-17F transcripts, formation of heterodimers, and the duIL-17F molecular weight were determined. Furthermore, expression profiles of duIL17F and duIL-17A were evaluated in mitogen-stimulated splenic lymphocytes and in several tissues from ducks infected with Salmonella Typhimurium, an intracellular bacterium and one of the causative agents of salmonellosis, which causes gastrointestinal tract inflammation and systemic infections.
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at 37 ◦ C. Tissue samples were collected at 0, 1, 4, 7 and 10 days post-infection. Control birds were orally inoculated with the same volume of phosphate buffered saline. Bacteria were recovered from the liver and spleen, and grown on xylose lysine deoxycholate agar plate (Difco, USA) for 24 h at 37 ◦ C. All animals were housed and reared according to the Guide for the Care and Use of Laboratory Animals established by Gyeongsang National University. 2.2. Molecular cloning of duIL-17F cDNA and sequence analysis Based on the sequence obtained from chicken IL-17F (Kim et al., 2012), the full-length duIL-17F was obtained from the cDNA of ConA-activated splenic lymphocytes by performing 5 /3 rapid amplification of cDNA ends (RACE) according to the manufacturer’s instructions using genespecific primers (for 5 RACE 5 -CAATGTTCAGGTCACTCCTT3 and for 3 RACE 5 -GACTTAATGGAGACTGCCAG-3 ) and high-fidelity DNA polymerase (Bioneer, Korea). The cloned cDNA in TA vector was confirmed by sequencing (Macrogen, Korea) and the nucleotide sequence was submitted to GenBank (KM035421). PCR was performed on a DNA Engine thermocycler (Bio-Rad, USA) with the standard conditions. Computer-assisted sequence analysis was performed for protein identification (http://www.expasy.org/ tools/), signal peptide sequence prediction (http://www. cbs.dtu.dk/services/SignaIP), homology analysis using MatGat software (Campanella et al., 2003) and phylogenetic tree analysis using MEGA program version (Tamura et al., 2011) and were bootstrapped 10,000 times. 2.3. Cell culture, plasmid construction and transfection COS-7 cells and primary splenic lymphocytes were cultured as previously described (Kim et al., 2014). The splenic lymphocytes were resuspended to 5 × 106 cells/ml and stimulated with 10 g/ml ConA (Amersham Bioscience, Sweden), 25 g/ml poly I:C, and 10 g/ml LPS from Escherichia coli, 0111:B4 (Sigma Aldrich, USA) for 0, 4, 8, and 24 h. The nucleotide sequence encoding duIL-17A and duIL-17F were amplified by PCR using the following specific primers: for duIL17A-myc or duIL17A-HA: forward 5 -GATCAAGCTTATGTCTCCAACCCTTCGTG-3 and reverse (myc) 5 -GATCGAATTCTTA
2. Materials and methods 2.1. Animals and infection Male Pekin ducklings (Anas platyrynchos) were purchased from Joowon ASTA Ducks (Gyeongnam, Korea) and raised in wire cages provided with feed and water ad libitum in a temperature controlled room. Constant light was provided for the duration of the experiments. The infected and non-infected ducks were separated in different buildings. Duck euthanasia was performed by cervical dislocation. Twenty five 2-week-old ducks were orally infected with 0.2 ml suspension of S. Typhimurium containing 4.4 × 108 colony-forming unit (CFU)/ml that were grown in Rappaport-Vassiliadis broth (Difco, USA) for 24 h
AGCCTGGTGCTGGATCAAG-3 GATCGAATTCTTA
or
reverse
(HA)
AGCCTGGTGCTGGATCAAG-3 , and for duIL-17F-myc: forward 5 -GATCAAGCTTATGGCTTTTGCCAGCTATG-3 and 5 -GATCGAATTCTTA AGCCTGGTGCTGGATCAAG-3 containing HindIII and EcoRI restriction enzyme sites (single underline) and myc or HA sequences (dotted line), respectively. PCR products were digested with HindIII and EcoRI, and cloned into the corresponding restriction enzyme sites of pcDNA 3.1 (Invitrogen, USA). The constructs and empty vector were transiently transfected to COS-7 cells using FuGENE
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Fig. 1. Molecular features of duIL-17F. (A) Nucleotide and deduced amino acid sequences of duIL-17F. The predicted signal region is underlined. The six conserved cysteine residues are highlighted with black boxes. The potential N-linked glycosylation site is boxed, the mRNA instability sequences in the 3 -noncoding region are in italics and underlined and the polyadenylation signal is in bold and underlined. (B) Phylogenetic analysis of avian, mammalian, and piscine IL-17A and IL-17F. The tree was constructed by the neighbor-joining tree method using amino acid sequences aligned with ClustalW2. GenBank accession numbers used in the comparison are NP 443104.1 (human IL-17F), NP 001015011.1 (rat IL-17F), NP 665855.2 (mouse IL-17F), NP 001179011.1 (bovine IL-17F), XP 001924401.3 (swine IL-17F), AFI61905.1 (chicken IL-17F), ABY68458.1 (duck IL-17A), AFG26505.1 (goose IL-17A), KM035421 (duck IL17F), AAC50341.1 (human IL-17A), NP 001100367.1 (rat IL-17A), AAB05222.1 (mouse IL-17A), CAD38489.1 (chicken IL-17A), BAD52431.1 (swine IL-17A), AAQ03220.1 (bovine IL-17A), NP 001118091.1 (trout IL-17A), NP 001018623.1 (ZebIL-17A/F1), NP 001018634.1 (ZebIL-17A/F2), NP 001018626.1 (ZebIL17A/F3), BAI82578.1 (FuguIL-17A/F1), BAI82579.1 (FuguIL-17A/F2), BAI82580.1 (FuguIL-17A/F3), NP 001191714.1 (MedakaIL-17A/F1), NP 001191715.1 (MedakaIL-17A/F3), NP 001191713.1 (MedakaIL-17A/F2), AFV61509.1 (TurbotIL-17A/F). (C) Schematic diagram of exon/intron structure of avian, mammalian, and piscine IL-17F genes and duck IL-17A. The thin line represents the intron and the black boxes indicate the exons of the respective genes. White boxes indicate UTR regions. GenBank accession numbers used in the comparison are NW 004676608.1 (duck IL-17A and duck IL-17F), NC 006090.3 (chicken IL-17F), NC 000006.12 (human IL-17F), NC 000067.6 (mouse IL-17F), NC 010449.4 (swine IL-17F), NC 005108.3 (rat IL-17F) and AC 000180.1 (bovine IL-17F).
6 transfection reagent (Promega, USA) following the manufacturer’s instructions.
buffer, boiled with sample buffer, separated by SDS-PAGE, and analyzed by Western blot.
2.4. Western blot analysis and Immunoprecipitation
2.5. Cytokine induction assay and quantitative real-time RT-PCR
Cells were lysed and Western blot analysis was performed as previously described (Kim et al., 2014). For immunoprecipitation, supernatants from duIL-17A-HA or duIL-17F-myc transfected COS-7 cells were mixed and incubated with either anti-myc, clone 9B11 (Cell Signaling, USA) or anti-HA antibody, clone 6E2 (Cell Signaling) with gentle shaking overnight at 4 ◦ C. Protein A agarose beads (Cell Signaling, USA) were added to the mixture and incubated with gentle shaking for 1 h at 4 ◦ C. After incubation, beads were washed five times with non-denaturing lysis
Total RNA was obtained from normal tissues, mitogenstimulated splenic lymphocytes, DEFs, and tissues pooled from three ducks infected with S. Typhimurium. Total RNA was extracted by a total RNA isolation solution, RiboEx (GeneAll, Korea) from samples homogenized with a grinder (Dalhan Sci. Korea) for tissues or with vortex for cells according to manufacturer’s instruction, purified with RNeasy Mini kit (Qiagen, Germany), and stored at −70 ◦ C. To remove any contaminating genomic DNA,
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samples were treated with DNase 1 (Thermo Scientific, USA). Single stranded cDNA was synthesized with QuantiTect reverse transcription kit (Qiagen) from the total RNA using random hexamer primers. Expression levels of duIL17A and duIL-17F were determined using the following primers: duIL-17A; forward 5 -ATGTCTCCAACCCTTCGT3 , reverse 5 -CCGTATCACCTTCCCGTA-3 and duIL-17F; forward 5 -CTGAGAGACTTAATGGAGACTG-3 , reverse 5 AGAATCTGAACGGCTGATG-3 . IFN-␣, IL-1, IL-6, IL-8 primers (Wei et al., 2014a) were used to evaluate the biological activity of duIL-17F and duIL-17A. Real-time RT-PCR was performed in duplicate using a CFX96 real-time PCR system (Bio-Rad, USA). To verify the presence of a single amplification product without primer dimers, a melting curve was obtained at the end of each run. Gene expression levels were quantified using the ct method with -actin (De Boever et al., 2008) as a reference for normalization.
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A
B
2.6. Statistical analysis Gathered data were statistically analyzed using the Student’s t-test or one-way ANOVA using InStat® software (GraphPad, USA). Differences were considered significant at P < 0.05. Data are expressed as mean ± standard error (SE). 3. Results and discussion 3.1. Cloning and characterization of duIL-17F The full-length cDNA of duIL-17F was cloned from ConA-activated splenic lymphocytes. The cloned duIL-17F cDNA was approximately 1.4 kb in length and contained a 63-bp 5 -UTR, a 501-bp ORF, which is predicted to encode 166 amino acid residues, and a 613-bp 3 -UTR. The 3 -UTR contained six AU-rich (ATTTA) sequences and a single consensus AATAAA polyadenylation signal. Computer-assisted sequence analysis revealed a 26 amino acid signal peptide, a single N-linked glycosylation site, and six cysteine residues involved in interchain disulfide bond formation that are conserved in the Th17 family (Starnes et al., 2001; Moseley et al., 2003) (Fig. 1A). This protein has a predicted molecular weight of 18.6 kDa (non-glycosylated) and a calculated isoelectric point of 9.81. Amino acid comparisons showed that duIL-17F shares 53.5% amino acid sequence identity with duIL-17A (Yoo et al., 2009), 77.5% with the chIL-17F (Kim et al., 2012) and 37–46% with corresponding mammalian homologues. Moreover, in a phylogenetic tree, duIL-17F formed a branch with chIL-17F in the same cluster as avian IL-17A (Fig. 1B) (Min and Lillehoj, 2002; Yoo et al., 2009; Wei et al., 2014b). The exon/intron organization of duIL-17F was quite similar to the chicken, mammalian, and piscine homologues (Fig. 1C). 3.2. Distribution of duIL-17F expression in unstimulated cells and in mitogen-stimulated lymphocytes The duIL-17F transcripts were expressed in a wide range of untreated tissues; levels were highest in the liver and moderate in the thymus, bursa, kidney, and intestinal tissues (Fig. 2A). This result is somewhat consistent
Fig. 2. mRNA expression levels of duIL-17A and duIL-17F in normal tissues and in mitogen-stimulated splenic lymphocytes. (A) Total RNA was extracted from various tissues from 2-week-old healthy ducks. Tissue samples were pooled from five ducks and subjected to real-time RT-PCR analysis. The mRNA expression levels were normalized to those of actin. Data represent the mean ± standard error from two independent experiments. (B) Splenic lymphocytes of 2-week-old healthy ducks were activated with 10 g/ml ConA, 10 g/ml LPS, or 25 g/ml poly I:C for the indicated times. The mRNA levels were normalized to those of actin. Data represent the mean ± standard error from three independent experiments with similar pattern results. P < 0.05 (*) or P < 0.01 (**) indicate significant differences in duIL-17A levels and P < 0.01 (††) indicate significant differences in duIL-17F levels as compared to unstimulated lymphocytes.
with the expression profiles of chIL-17F we described in a previous study (Kim et al., 2012). Other studies also reported that expression of these two cytokines differs in various tissues (Kawaguchi et al., 2001; Gomez-Rodriguez et al., 2009). In mitogen-stimulated splenic lymphocytes (Fig. 2B), although expression levels of duIL-17F were lower than duIL-17A levels, both duIL-17A and duIL-17F transcripts were generally up-regulated in ConA- and LPS-activated splenic lymphocytes in a time-dependent manner but not in poly I:C stimulated cells compared to unstimulated cells (Fig. 2B). Interestingly, in previous studies, duck embryonic fibroblasts (DEFs) stimulated with poly I:C showed unchanged or decreased Th17-promoting cytokine, IL-1, and IL-6 transcripts (Zhao et al., 2013). However, poly I:C stimulation of chicken splenic lymphocytes and an epithelial cell line shows significant increases in IL-17A and IL-17F, and IL-8 expression, respectively (Esnault et al., 2011; Kim et al., 2012).
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3.3. Molecular weight and biological activity of duIL-17F Constructs containing either myc-tagged duIL-17F or duIL-17A were transfected into COS-7 cells. The molecular weight bands of recombinant duIL-17F protein were detected at approximately 20 kDa in both culture supernatants and in cell lysates; the observed molecular weight was very similar to that of duIL-17A (Fig. 3A) and to those of chIL-17A and chIL-17F (Kim et al., 2012). To assay the effect of recombinant duIL-17F expression on the production of cytokines in primary DEFs, DEFs were incubated for 6 h with the supernatants from COS-7 cells transfected with duIL-17F. The duIL-17F and duIL-17A treatments led to increased expression of IL-8, IL-1, and IL-6, but not IFN-␣ (Fig. 3B). 3.4. Interaction between duIL-17A and duIL-17F Generally, IL-17F forms homodimers or heterodimers with IL-17A and functions through engagement of the IL-17 receptor (IL-17R) (Iwakura et al., 2011). Thus, we used co-immunoprecipitation experiments to determine whether duIL-17F interacts with duIL-17A. Supernatants of duIL-17F-myc tranfected COS-7 were incubated with supernatants of duIL-17A-HA transfected COS-7 and analyzed using Western blotting. The observed interaction did not appear to be due to non-specific interaction with protein A beads, myc, or HA proteins (Fig. 3C, lanes 1–3), while duIL-17F elution with duIL-17A (Fig. 3C, lane 4), suggest that duIL-17F directly interacts with duIL-17A. This finding is consistent with the ability of IL-17F to form a heterodimer with IL-17A in other species (Wright et al., 2007). 3.5. Quantitative analysis of duIL-17A and duIL-17F mRNA expression levels in Salmonella-infected ducks Although several studies have demonstrated the biological roles of Th17 cytokines in different animal and disease models, only a few studies have investigated the roles of Th17 cytokines, particularly IL-17A and IL-17F, in host defense against Salmonella species, an important zoonotic pathogen (Schulz et al., 2008). Generally in systemic infections, Salmonella enters the intestinal organs, especially the jejunum, ileum, ceca, and cecal tonsils and is carried to other organs such as the spleen and liver
Fig. 3. Biological activity and molecular weight of duIL-17F. (A) Detection of duIL-17F protein by Western blot analysis. COS-7 cells were transiently transfected with duIL-17A-myc, duIL-17F-myc, or vector pcDNA 3.1. Supernatants and cell lysates were collected after 48 h and separated by SDS-PAGE under reducing conditions. Arrow indicates specific bands. Data are one representative experiment of two independent experiments with similar pattern results. (B) Induction of proinflammatory cytokines in primary DEFs. Primary DEFs were obtained from 10-day-old embryos as previously described (Kim et al., 2012), stimulated with the conditioned medium from COS-7 cells transfected with duIL-17A, duIL-17F,
or empty vector (pcDNA 3.1) for 6 h at 41 ◦ C in 5% CO2 , and analyzed by real-time RT-PCR. Expression levels were normalized to those of -actin. Data represent the mean ± standard error from three independent experiments with similar pattern results. ** and †† (P < 0.01) indicate significant differences in duIL-17F and duIL-17A levels respectively, as compared to empty vector. The inset shows enlargement of IL-6, IL-1 and IFN-␣ mRNA expression levels. (C) Heterodimer formation between HA-tagged duIL-17A and myc-tagged duIL-17F. Supernatants from duIL-17A-HA or duIL-17F-myc transfected COS-7 cells were mixed and incubated with either anti-myc or anti-HA antibody. The complex was precipitated using protein A-agarose beads. The precipitate was separated using SDS-PAGE under reducing conditions with the corresponding antibodies (rows c and d). The non-immunoprecipitated samples were blotted as loading controls (rows a and b). Data are one representative experiment of two independent experiments with similar pattern results. IP, immunoprecipitation; WB, Western blotting; HA, hemagglutinin; myc, c-myc.
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Fig. 4. mRNA expression profiles of duIL-17A and duIL-17F in Salmonella-infected ducks. IL-17A and IL-17F mRNA expression was quantified by real-time RT-PCR in the liver (A), spleen (B), and cecal-tonsil (C). The mRNA levels were normalized to those of -actin. Data represent the mean ± standard error from three independent experiments with similar pattern results. * (P < 0.05) indicates significant differences as compared to duIL-17A levels in uninfected ducks. (D) Bacterial load of S. Typhimurium in the liver and spleen. Three ducks were sacrificed at each time point and the liver and spleen were aseptically collected for bacterial recovery. Error bars represent mean ± standard errors of three samples. Data are one representative experiment of two independent experiments with similar pattern results. dpi, days post-infection.
(Kaiser et al., 2000). In an initial effort to determine expression patterns of duIL-17F transcript, ducks were infected orally with S. Typhimurium. Compared to uninfected ducks, expression levels of duIL-17F transcript were unchanged in the liver and spleen. duIL-17A levels, however, were elevated in the liver on day 7 and in the spleen on day 4 post-infection (Fig. 4A and B). Similarly, IL-17A expression is increased in the spleen of Lohmann and ISA Brown chickens intravenously infected with Salmonella Enteritidis (Matulova et al., 2012). Expression levels of the IL-17A and IL-17F are known to be elevated on day 4 and 7 in the spleen and liver of chickens infected with Salmonella gallinarum, but not in chickens infected with S. Typhimurium, which display an increased IL-17A and IL-17F expression in the spleen on day 7 post-infection (Kim et al., 2014). Interestingly, IL-17A knockout mice challenged with S. Enteritidis display increased bacterial burden in the spleen and liver compared with uninfected mice (Schulz et al., 2008). Salmonella spp. colonize most specifically the ceca and cecal tonsils (Crhanova et al., 2011) and S. Typhimurium infection induces proinflammatory cytokines IL-1 and IL-6 in the intestinal tissues of chickens (Fasina et al., 2008). We therefore monitored duIL-17A and duIL-17F transcript levels in the cecal tonsil of Salmonella-infected ducks. Expression levels of duIL-17A and duIL-17F transcripts were statistically unchanged in the cecal tonsils (Fig. 4C) and cecum (data not shown) in S. Typhimuriuminfected ducks as compared to the uninfected ducks. It
is interesting to note that IL-17A mRNA expression is unchanged in the cecum of chickens orally infected with S. Enteritidis (Matulova et al., 2012), and significantly downregulated in the ileum mucosa of pigs orally infected with S. Typhimurium (Collado-Romero et al., 2012). By contrast, increased expression of IL-17A transcript is detected in the cecum of chickens orally infected with S. Enteritidis (Crhanova et al., 2011), and in the ceca of streptomycinpretreated mice orally infected with S. Typhimurium (Godinez et al., 2008). Generally, expression levels of duIL17A and duIL-17F mRNA in Salmonella-infected ducks were weakly increased compared to Salmonella-infected chickens (Kim et al., 2014). We therefore monitored S. Typhimurium infection in the spleen and liver postinfection, and were able to identify bacteria in both organs in infected ducks (Fig. 4D). Although Salmonella infections in ducks and chickens are probably the most important source of Salmonellaassociated foodborne diseases in human, basic information about Salmonella control in duck industry depends mainly on research on salmonellosis in chickens, which may not be directly transferable to ducks. We cloned duIL17F and investigated expression patterns and functions. In Salmonella-infected ducks duIL-17A and duIL-17F levels were unchanged or weakly elevated in several tissues tested in contrast to Salmonella-infected chickens. Taken together, these findings suggest that mRNA expression levels of IL-17A and IL-17F in response to Salmonella infection
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Kawaguchi, M., Onuchic, L.F., Li, X.D., Essayan, D.M., Schroeder, J., Xiao, H.Q., Liu, M.C., Krishnaswamy, G., Germino, G., Huang, S.K., 2001. Identification of a novel cytokine, ML-1, and its expression in subjects with asthma. J. Immunol. 167, 4430–4435. Kim, W.H., Jeong, J., Park, A.R., Yim, D., Kim, Y.H., Kim, K.D., Chang, H.H., Lillehoj, H.S., Lee, B.H., Min, W., 2012. Chicken IL-17F: Identification and comparative expression analysis in Eimeria-infected chickens. Dev. Comp. Immunol. 38, 401–409. Kim, W.H., Jeong, J., Park, A.R., Yim, D., Kim, S., Chang, H.H., Yang, S.H., Kim, D.H., Lillehoj, H.S., Min, W., 2014. Downregulation of chicken interleukin-17 receptor A during Eimeria infection. Infect. Immun. 82, 3845–3854. Leppkes, M., Becker, C., Ivanov, I.I., Hirth, S., Wirtz, S., Neufert, C., Pouly, S., Murphy, A.J., Valenzuela, D.M., Yancopoulos, G.D., Becher, B., Littman, D.R., Neurath, M.F., 2009. ROR␥-expressing Th17 cells induce murine chronic intestinal inflammation via redundant effects of IL-17A and IL-17F. Gastroenterology 136, 257–267. Matulova, M., Havlickova, H., Sisak, F., Rychlik, I., 2012. Vaccination of chickens with Salmonella pathogenicity island (SPI) 1 and SPI2 defective mutants of Salmonella enterica serovar Enteritidis. Vaccine 30, 2090–2097. Min, W., Lillehoj, H.S., 2002. Isolation and characterization of chicken interleukin-17 cDNA. J. Interferon Cytokine Res. 22, 1123–1128. Min, W., Kim, W.H., Lillehoj, E.P., Lillehoj, H.S., 2013. Recent progress in host immunity to avian coccidiosis: IL-17 family cytokines as sentinels of the intestinal mucosa. Dev. Comp. Immunol. 41, 418–428. Moseley, T.A., Haudenschild, D.R., Rose, L., Reddi, A.H., 2003. Interleukin17 family and IL-17 receptors. Cytokine Growth Factor Rev. 14, 155–174. Oda, N., Canelos, P.B., Essayan, D.M., Plunkett, B.A., Myers, A.C., Huang, S.K., 2005. Interleukin-17F induces pulmonary neutrophilia and amplifies antigen-induced allergic response. Am. J. Respir. Crit. Care Med. 171, 12–18. O’Sullivan, T., Konefka, R.S., Gross, E., Tran, M., Mayfield, S.P., Ikeda, H., Bui, J.D., 2014. Interleukin-17D mediates tumor rejection through recruitment of natural killer cells. Cell. Rep. 7, 989–998. Schulz, S.M., Kholer, G., Holscher, C., Iwakura, Y., Alber, G., 2008. IL-17A is produced by Th17, ␥␦ T cells and other CD4− lymphocytes during infection with Salmonella enterica serovar enteritidis and has a mild effect in bacterial clearance. Infect. Immunol. 20, 1129–1138. Staeheli, P., Puehler, F., Schneider, K., Gobel, T.W., Kaspers, B., 2001. Cytokines of birds: conserved functions – a largely different look. J. Interferon Cytokine Res. 21, 993–1010. Starnes, T., Robertson, M.J., Sledge, G., Kelich, S., Nakshatri, H., Broxmeyer, H.E., Hromas, R., 2001. Cutting edge: IL-17F, a novel cytokine selectively expressed in activated T cells and monocytes, regulates angiogenesis and endothelial cell cytokine production. J. Immunol. 167, 4137–4140. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Wei, L., Cui, J., Song, Y., Zhang, S., Han, F., Yuan, R., Gong, L., Jiao, P., Liao, M., 2014a. Duck MDA5 functions in innate immunity against H5N1 highly pathogenic avian influenza virus infections. Vet. Res. 45, 66. Wei, S., Liu, X., Gao, M., Zhang, W., Zhu, Y., Ma, B., Wang, J., 2014b. Cloning and characterization of goose interleukin-17A cDNA. Res. Vet. Sci. 96, 118–123. Wright, J.F., Bennett, F., Li, B., Brooks, J., Luxenberg, D.P., Whitters, M.J., Tomkinson, K.N., Fitz, L.J., Wolfman, N.M., Collins, M., Joannapoulos, K.D., Kishore, M.C., Carreno, B.M., 2007. The human IL-17F/IL-17A heterodimeric cytokine signals through the IL-17RA/IL-17RC receptor complex. J. Immunol. 181, 2799–2805. Xie, Y., Sheng, W., Xiang, J., Ye, Z., Yang, J., 2010. Interleukin-17F suppresses hepatocarcinoma cell growth via inhibition of tumor angiogenesis. Cancer Investig. 28, 598–607. Yang, X.O., Chang, S.H., Park, H., Nurieva, R., Shah, B., Acero, L., Wang, Y.H., Schluns, K.S., Broaddus, R.R., Zhu, Z., Dong, C., 2008. Regulation of inflammatory responses by IL-17F. J. Exp. Med. 205, 1063–1075. Yoo, J., Jang, S.I., Kim, S., Cho, J.H., Lee, H.J., Rhee, M.H., Lillehoj, H.S., Min, W., 2009. Molecular characterization of duck interleukin-17. Vet. Immunol. Immunopathol. 132, 318–322. Zhao, W., Huang, Z., Chen, Y., Zhang, Y., Rong, G., Mu, C., Xu, Q., Chen, G., 2013. Molecular cloning and functional analysis of the duck TLR4 gene. Int. J. Mol. Sci. 14, 18615–18628.
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Short communication
Molecular cloning, characterization and mRNA expression of duck interleukin-17F Woo H. Kim a,1 , Cherry P. Fernandez a,1 , Joyce Anne R. Diaz a , Jipseol Jeong b , Suk Kim a , Hyun S. Lillehoj c , Hong H. Chang d , Wongi Min a,∗ a b c d
College of Veterinary Medicine & Institute of Animal Medicine, Gyeongsang National University, Jinju 660-701, Republic of Korea Environmental Health Research Department, Environmental Research Complex, Incheon 404-708, Republic of Korea Animal Biosciences and Biotechnology Laboratory, BARC, ARS, USDA, Beltsville, MD 20705, USA Department of Animal Science, College of Agriculture, Gyeongsang National University, Jinju 660-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 11 November 2014 Received in revised form 7 February 2015 Accepted 19 February 2015 Keywords: Duck Cytokine Salmonella infection Interleukin-17F
a b s t r a c t Interleukin-17F (IL-17F) is a proinflammatory cytokine that plays an important role in gut homeostasis. A full-length duck IL-17F (duIL-17F) cDNA with a 510-bp coding region was identified in ConA-activated splenic lymphocytes. duIL-17F is predicted to encode 166 amino acids, including a 26-amino acid signal peptide, a single N-linked glycosylation site, and six cysteine residues that are conserved in mammalian IL-17. duIL-17F shares 77.5% amino acid sequence identity with chicken IL-17F (chIL-17F), 37–46% with corresponding mammalian homologues, and 53.5% with the previously described duck IL-17A (duIL-17A). The duIL-17F transcripts were expressed in a wide range of untreated tissues; levels were highest in the liver and moderate in the thymus, bursa, kidney, and intestinal tissues. Expression levels of duIL-17F transcript were slightly up-regulated in ConA- and LPS-activated splenic lymphocytes but not in poly I:C stimulated cells. duIL17F forms heterodimers with duIL-17A. Recombinant duIL-17F, like duIL-17A, induced IL-1, IL-6, and IL-8 expression in duck embryonic fibroblasts (DEFs). duIL-17A, but not duIL-17F expression, was significantly up-regulated in the liver and spleen of Salmonella Typhimurium-infected ducks. Further analysis of the contributions of IL-17F to different Salmonella spp. or other disease models will be required to expand our understanding of its biological functions. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The IL-17 family consists of six cytokine members (IL17A to IL-17F), which have been shown to have varying biological roles and degrees of intermolecular amino acid
∗ Corresponding author at: College of Veterinary Medicine, Gyeongsang National University, 501 Jinju-daero, Jinju, Gyeongnam 660-701, Republic of Korea. Tel.: +82 55 772 2357; fax: +82 55 772 2349. E-mail address: [email protected] (W. Min). 1 Woo H. Kim and Cherry P. Fernandez contributed equally to this work. http://dx.doi.org/10.1016/j.vetimm.2015.02.007 0165-2427/© 2015 Elsevier B.V. All rights reserved.
sequence homology in mammals. These six IL-17-related cytokines form homodimers or heterodimers and generally function through engagement of the IL-17 receptor (IL-17R) family, which includes five molecules (IL-17RA to IL-17RE) (Iwakura et al., 2011). IL-17 cytokines play important roles in host defense against variety of pathogens by inducing the generation of proinflammatory cytokines and chemokines, and have been implicated in autoimmune diseases, inflammatory diseases, and tumors (Iwakura et al., 2011; O’Sullivan et al., 2014). The best-studied members of the IL-17 family are IL-17A and IL-17F which share high amino acid sequence identity (approximately 50%)
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and bind the same receptors. Thus, these two cytokines are likely to have similar and/or overlapping biological activities (Moseley et al., 2003; Min et al., 2013). Although they have been shown to have some similar functions, IL17A and IL-17F have been suggested by some studies to be differentially regulated and to have their own distinct activities (Yang et al., 2008; Ishigame et al., 2009). IL-17F was initially identified in activated T cells and monocytes using the human IL-17A genomic sequence (Starnes et al., 2001). Unlike IL-17A, IL-17F is generally expressed in a variety of cell types and tissues, suggesting that IL-17F is involved in a broader range of biological functions (Gomez-Rodriguez et al., 2009; Kim et al., 2012). Antiangiogenic function of IL-17F has been described in endothelial cells and hepatocarcinoma cells (Starnes et al., 2001; Xie et al., 2010) and defective expression of IL-17F has been implicated in bronchial asthma (Kawaguchi et al., 2001), antigen-induced allergic response (Oda et al., 2005), chronic obstructive pulmonary disease (Hizawa et al., 2006), psoriasis (Fujishima et al., 2010), chronic intestinal inflammation (Leppkes et al., 2009), and rheumatoid arthritis (Hurst et al., 2002). The avian immune system provides an important model for the study of basic and applied immunology. Due to the general lack of cross-reactivity and the low level of sequence conservation between avian and mammalian cytokines (Staeheli et al., 2001; Starnes et al., 2001), identification of immune-related genes has been challenging. The recent dramatic growth of the duck industry is anticipated to increase the occurrence of diseases including Salmonella infection (Cha et al., 2013). Recently, duIL-17A was identified in ConA-activated splenic lymphocytes (Yoo et al., 2009) leading us to become interested in identify and characterize the duIL-17F. In this study, we identified a full-length duIL-17F cDNA. Through reverse transcription quantitative real-time RT-PCR and Western blot analysis, the tissue distribution of duIL-17F transcripts, formation of heterodimers, and the duIL-17F molecular weight were determined. Furthermore, expression profiles of duIL17F and duIL-17A were evaluated in mitogen-stimulated splenic lymphocytes and in several tissues from ducks infected with Salmonella Typhimurium, an intracellular bacterium and one of the causative agents of salmonellosis, which causes gastrointestinal tract inflammation and systemic infections.
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at 37 ◦ C. Tissue samples were collected at 0, 1, 4, 7 and 10 days post-infection. Control birds were orally inoculated with the same volume of phosphate buffered saline. Bacteria were recovered from the liver and spleen, and grown on xylose lysine deoxycholate agar plate (Difco, USA) for 24 h at 37 ◦ C. All animals were housed and reared according to the Guide for the Care and Use of Laboratory Animals established by Gyeongsang National University. 2.2. Molecular cloning of duIL-17F cDNA and sequence analysis Based on the sequence obtained from chicken IL-17F (Kim et al., 2012), the full-length duIL-17F was obtained from the cDNA of ConA-activated splenic lymphocytes by performing 5 /3 rapid amplification of cDNA ends (RACE) according to the manufacturer’s instructions using genespecific primers (for 5 RACE 5 -CAATGTTCAGGTCACTCCTT3 and for 3 RACE 5 -GACTTAATGGAGACTGCCAG-3 ) and high-fidelity DNA polymerase (Bioneer, Korea). The cloned cDNA in TA vector was confirmed by sequencing (Macrogen, Korea) and the nucleotide sequence was submitted to GenBank (KM035421). PCR was performed on a DNA Engine thermocycler (Bio-Rad, USA) with the standard conditions. Computer-assisted sequence analysis was performed for protein identification (http://www.expasy.org/ tools/), signal peptide sequence prediction (http://www. cbs.dtu.dk/services/SignaIP), homology analysis using MatGat software (Campanella et al., 2003) and phylogenetic tree analysis using MEGA program version (Tamura et al., 2011) and were bootstrapped 10,000 times. 2.3. Cell culture, plasmid construction and transfection COS-7 cells and primary splenic lymphocytes were cultured as previously described (Kim et al., 2014). The splenic lymphocytes were resuspended to 5 × 106 cells/ml and stimulated with 10 g/ml ConA (Amersham Bioscience, Sweden), 25 g/ml poly I:C, and 10 g/ml LPS from Escherichia coli, 0111:B4 (Sigma Aldrich, USA) for 0, 4, 8, and 24 h. The nucleotide sequence encoding duIL-17A and duIL-17F were amplified by PCR using the following specific primers: for duIL17A-myc or duIL17A-HA: forward 5 -GATCAAGCTTATGTCTCCAACCCTTCGTG-3 and reverse (myc) 5 -GATCGAATTCTTA
2. Materials and methods 2.1. Animals and infection Male Pekin ducklings (Anas platyrynchos) were purchased from Joowon ASTA Ducks (Gyeongnam, Korea) and raised in wire cages provided with feed and water ad libitum in a temperature controlled room. Constant light was provided for the duration of the experiments. The infected and non-infected ducks were separated in different buildings. Duck euthanasia was performed by cervical dislocation. Twenty five 2-week-old ducks were orally infected with 0.2 ml suspension of S. Typhimurium containing 4.4 × 108 colony-forming unit (CFU)/ml that were grown in Rappaport-Vassiliadis broth (Difco, USA) for 24 h
AGCCTGGTGCTGGATCAAG-3 GATCGAATTCTTA
or
reverse
(HA)
AGCCTGGTGCTGGATCAAG-3 , and for duIL-17F-myc: forward 5 -GATCAAGCTTATGGCTTTTGCCAGCTATG-3 and 5 -GATCGAATTCTTA AGCCTGGTGCTGGATCAAG-3 containing HindIII and EcoRI restriction enzyme sites (single underline) and myc or HA sequences (dotted line), respectively. PCR products were digested with HindIII and EcoRI, and cloned into the corresponding restriction enzyme sites of pcDNA 3.1 (Invitrogen, USA). The constructs and empty vector were transiently transfected to COS-7 cells using FuGENE
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Fig. 1. Molecular features of duIL-17F. (A) Nucleotide and deduced amino acid sequences of duIL-17F. The predicted signal region is underlined. The six conserved cysteine residues are highlighted with black boxes. The potential N-linked glycosylation site is boxed, the mRNA instability sequences in the 3 -noncoding region are in italics and underlined and the polyadenylation signal is in bold and underlined. (B) Phylogenetic analysis of avian, mammalian, and piscine IL-17A and IL-17F. The tree was constructed by the neighbor-joining tree method using amino acid sequences aligned with ClustalW2. GenBank accession numbers used in the comparison are NP 443104.1 (human IL-17F), NP 001015011.1 (rat IL-17F), NP 665855.2 (mouse IL-17F), NP 001179011.1 (bovine IL-17F), XP 001924401.3 (swine IL-17F), AFI61905.1 (chicken IL-17F), ABY68458.1 (duck IL-17A), AFG26505.1 (goose IL-17A), KM035421 (duck IL17F), AAC50341.1 (human IL-17A), NP 001100367.1 (rat IL-17A), AAB05222.1 (mouse IL-17A), CAD38489.1 (chicken IL-17A), BAD52431.1 (swine IL-17A), AAQ03220.1 (bovine IL-17A), NP 001118091.1 (trout IL-17A), NP 001018623.1 (ZebIL-17A/F1), NP 001018634.1 (ZebIL-17A/F2), NP 001018626.1 (ZebIL17A/F3), BAI82578.1 (FuguIL-17A/F1), BAI82579.1 (FuguIL-17A/F2), BAI82580.1 (FuguIL-17A/F3), NP 001191714.1 (MedakaIL-17A/F1), NP 001191715.1 (MedakaIL-17A/F3), NP 001191713.1 (MedakaIL-17A/F2), AFV61509.1 (TurbotIL-17A/F). (C) Schematic diagram of exon/intron structure of avian, mammalian, and piscine IL-17F genes and duck IL-17A. The thin line represents the intron and the black boxes indicate the exons of the respective genes. White boxes indicate UTR regions. GenBank accession numbers used in the comparison are NW 004676608.1 (duck IL-17A and duck IL-17F), NC 006090.3 (chicken IL-17F), NC 000006.12 (human IL-17F), NC 000067.6 (mouse IL-17F), NC 010449.4 (swine IL-17F), NC 005108.3 (rat IL-17F) and AC 000180.1 (bovine IL-17F).
6 transfection reagent (Promega, USA) following the manufacturer’s instructions.
buffer, boiled with sample buffer, separated by SDS-PAGE, and analyzed by Western blot.
2.4. Western blot analysis and Immunoprecipitation
2.5. Cytokine induction assay and quantitative real-time RT-PCR
Cells were lysed and Western blot analysis was performed as previously described (Kim et al., 2014). For immunoprecipitation, supernatants from duIL-17A-HA or duIL-17F-myc transfected COS-7 cells were mixed and incubated with either anti-myc, clone 9B11 (Cell Signaling, USA) or anti-HA antibody, clone 6E2 (Cell Signaling) with gentle shaking overnight at 4 ◦ C. Protein A agarose beads (Cell Signaling, USA) were added to the mixture and incubated with gentle shaking for 1 h at 4 ◦ C. After incubation, beads were washed five times with non-denaturing lysis
Total RNA was obtained from normal tissues, mitogenstimulated splenic lymphocytes, DEFs, and tissues pooled from three ducks infected with S. Typhimurium. Total RNA was extracted by a total RNA isolation solution, RiboEx (GeneAll, Korea) from samples homogenized with a grinder (Dalhan Sci. Korea) for tissues or with vortex for cells according to manufacturer’s instruction, purified with RNeasy Mini kit (Qiagen, Germany), and stored at −70 ◦ C. To remove any contaminating genomic DNA,
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samples were treated with DNase 1 (Thermo Scientific, USA). Single stranded cDNA was synthesized with QuantiTect reverse transcription kit (Qiagen) from the total RNA using random hexamer primers. Expression levels of duIL17A and duIL-17F were determined using the following primers: duIL-17A; forward 5 -ATGTCTCCAACCCTTCGT3 , reverse 5 -CCGTATCACCTTCCCGTA-3 and duIL-17F; forward 5 -CTGAGAGACTTAATGGAGACTG-3 , reverse 5 AGAATCTGAACGGCTGATG-3 . IFN-␣, IL-1, IL-6, IL-8 primers (Wei et al., 2014a) were used to evaluate the biological activity of duIL-17F and duIL-17A. Real-time RT-PCR was performed in duplicate using a CFX96 real-time PCR system (Bio-Rad, USA). To verify the presence of a single amplification product without primer dimers, a melting curve was obtained at the end of each run. Gene expression levels were quantified using the ct method with -actin (De Boever et al., 2008) as a reference for normalization.
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A
B
2.6. Statistical analysis Gathered data were statistically analyzed using the Student’s t-test or one-way ANOVA using InStat® software (GraphPad, USA). Differences were considered significant at P < 0.05. Data are expressed as mean ± standard error (SE). 3. Results and discussion 3.1. Cloning and characterization of duIL-17F The full-length cDNA of duIL-17F was cloned from ConA-activated splenic lymphocytes. The cloned duIL-17F cDNA was approximately 1.4 kb in length and contained a 63-bp 5 -UTR, a 501-bp ORF, which is predicted to encode 166 amino acid residues, and a 613-bp 3 -UTR. The 3 -UTR contained six AU-rich (ATTTA) sequences and a single consensus AATAAA polyadenylation signal. Computer-assisted sequence analysis revealed a 26 amino acid signal peptide, a single N-linked glycosylation site, and six cysteine residues involved in interchain disulfide bond formation that are conserved in the Th17 family (Starnes et al., 2001; Moseley et al., 2003) (Fig. 1A). This protein has a predicted molecular weight of 18.6 kDa (non-glycosylated) and a calculated isoelectric point of 9.81. Amino acid comparisons showed that duIL-17F shares 53.5% amino acid sequence identity with duIL-17A (Yoo et al., 2009), 77.5% with the chIL-17F (Kim et al., 2012) and 37–46% with corresponding mammalian homologues. Moreover, in a phylogenetic tree, duIL-17F formed a branch with chIL-17F in the same cluster as avian IL-17A (Fig. 1B) (Min and Lillehoj, 2002; Yoo et al., 2009; Wei et al., 2014b). The exon/intron organization of duIL-17F was quite similar to the chicken, mammalian, and piscine homologues (Fig. 1C). 3.2. Distribution of duIL-17F expression in unstimulated cells and in mitogen-stimulated lymphocytes The duIL-17F transcripts were expressed in a wide range of untreated tissues; levels were highest in the liver and moderate in the thymus, bursa, kidney, and intestinal tissues (Fig. 2A). This result is somewhat consistent
Fig. 2. mRNA expression levels of duIL-17A and duIL-17F in normal tissues and in mitogen-stimulated splenic lymphocytes. (A) Total RNA was extracted from various tissues from 2-week-old healthy ducks. Tissue samples were pooled from five ducks and subjected to real-time RT-PCR analysis. The mRNA expression levels were normalized to those of actin. Data represent the mean ± standard error from two independent experiments. (B) Splenic lymphocytes of 2-week-old healthy ducks were activated with 10 g/ml ConA, 10 g/ml LPS, or 25 g/ml poly I:C for the indicated times. The mRNA levels were normalized to those of actin. Data represent the mean ± standard error from three independent experiments with similar pattern results. P < 0.05 (*) or P < 0.01 (**) indicate significant differences in duIL-17A levels and P < 0.01 (††) indicate significant differences in duIL-17F levels as compared to unstimulated lymphocytes.
with the expression profiles of chIL-17F we described in a previous study (Kim et al., 2012). Other studies also reported that expression of these two cytokines differs in various tissues (Kawaguchi et al., 2001; Gomez-Rodriguez et al., 2009). In mitogen-stimulated splenic lymphocytes (Fig. 2B), although expression levels of duIL-17F were lower than duIL-17A levels, both duIL-17A and duIL-17F transcripts were generally up-regulated in ConA- and LPS-activated splenic lymphocytes in a time-dependent manner but not in poly I:C stimulated cells compared to unstimulated cells (Fig. 2B). Interestingly, in previous studies, duck embryonic fibroblasts (DEFs) stimulated with poly I:C showed unchanged or decreased Th17-promoting cytokine, IL-1, and IL-6 transcripts (Zhao et al., 2013). However, poly I:C stimulation of chicken splenic lymphocytes and an epithelial cell line shows significant increases in IL-17A and IL-17F, and IL-8 expression, respectively (Esnault et al., 2011; Kim et al., 2012).
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3.3. Molecular weight and biological activity of duIL-17F Constructs containing either myc-tagged duIL-17F or duIL-17A were transfected into COS-7 cells. The molecular weight bands of recombinant duIL-17F protein were detected at approximately 20 kDa in both culture supernatants and in cell lysates; the observed molecular weight was very similar to that of duIL-17A (Fig. 3A) and to those of chIL-17A and chIL-17F (Kim et al., 2012). To assay the effect of recombinant duIL-17F expression on the production of cytokines in primary DEFs, DEFs were incubated for 6 h with the supernatants from COS-7 cells transfected with duIL-17F. The duIL-17F and duIL-17A treatments led to increased expression of IL-8, IL-1, and IL-6, but not IFN-␣ (Fig. 3B). 3.4. Interaction between duIL-17A and duIL-17F Generally, IL-17F forms homodimers or heterodimers with IL-17A and functions through engagement of the IL-17 receptor (IL-17R) (Iwakura et al., 2011). Thus, we used co-immunoprecipitation experiments to determine whether duIL-17F interacts with duIL-17A. Supernatants of duIL-17F-myc tranfected COS-7 were incubated with supernatants of duIL-17A-HA transfected COS-7 and analyzed using Western blotting. The observed interaction did not appear to be due to non-specific interaction with protein A beads, myc, or HA proteins (Fig. 3C, lanes 1–3), while duIL-17F elution with duIL-17A (Fig. 3C, lane 4), suggest that duIL-17F directly interacts with duIL-17A. This finding is consistent with the ability of IL-17F to form a heterodimer with IL-17A in other species (Wright et al., 2007). 3.5. Quantitative analysis of duIL-17A and duIL-17F mRNA expression levels in Salmonella-infected ducks Although several studies have demonstrated the biological roles of Th17 cytokines in different animal and disease models, only a few studies have investigated the roles of Th17 cytokines, particularly IL-17A and IL-17F, in host defense against Salmonella species, an important zoonotic pathogen (Schulz et al., 2008). Generally in systemic infections, Salmonella enters the intestinal organs, especially the jejunum, ileum, ceca, and cecal tonsils and is carried to other organs such as the spleen and liver
Fig. 3. Biological activity and molecular weight of duIL-17F. (A) Detection of duIL-17F protein by Western blot analysis. COS-7 cells were transiently transfected with duIL-17A-myc, duIL-17F-myc, or vector pcDNA 3.1. Supernatants and cell lysates were collected after 48 h and separated by SDS-PAGE under reducing conditions. Arrow indicates specific bands. Data are one representative experiment of two independent experiments with similar pattern results. (B) Induction of proinflammatory cytokines in primary DEFs. Primary DEFs were obtained from 10-day-old embryos as previously described (Kim et al., 2012), stimulated with the conditioned medium from COS-7 cells transfected with duIL-17A, duIL-17F,
or empty vector (pcDNA 3.1) for 6 h at 41 ◦ C in 5% CO2 , and analyzed by real-time RT-PCR. Expression levels were normalized to those of -actin. Data represent the mean ± standard error from three independent experiments with similar pattern results. ** and †† (P < 0.01) indicate significant differences in duIL-17F and duIL-17A levels respectively, as compared to empty vector. The inset shows enlargement of IL-6, IL-1 and IFN-␣ mRNA expression levels. (C) Heterodimer formation between HA-tagged duIL-17A and myc-tagged duIL-17F. Supernatants from duIL-17A-HA or duIL-17F-myc transfected COS-7 cells were mixed and incubated with either anti-myc or anti-HA antibody. The complex was precipitated using protein A-agarose beads. The precipitate was separated using SDS-PAGE under reducing conditions with the corresponding antibodies (rows c and d). The non-immunoprecipitated samples were blotted as loading controls (rows a and b). Data are one representative experiment of two independent experiments with similar pattern results. IP, immunoprecipitation; WB, Western blotting; HA, hemagglutinin; myc, c-myc.
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Fig. 4. mRNA expression profiles of duIL-17A and duIL-17F in Salmonella-infected ducks. IL-17A and IL-17F mRNA expression was quantified by real-time RT-PCR in the liver (A), spleen (B), and cecal-tonsil (C). The mRNA levels were normalized to those of -actin. Data represent the mean ± standard error from three independent experiments with similar pattern results. * (P < 0.05) indicates significant differences as compared to duIL-17A levels in uninfected ducks. (D) Bacterial load of S. Typhimurium in the liver and spleen. Three ducks were sacrificed at each time point and the liver and spleen were aseptically collected for bacterial recovery. Error bars represent mean ± standard errors of three samples. Data are one representative experiment of two independent experiments with similar pattern results. dpi, days post-infection.
(Kaiser et al., 2000). In an initial effort to determine expression patterns of duIL-17F transcript, ducks were infected orally with S. Typhimurium. Compared to uninfected ducks, expression levels of duIL-17F transcript were unchanged in the liver and spleen. duIL-17A levels, however, were elevated in the liver on day 7 and in the spleen on day 4 post-infection (Fig. 4A and B). Similarly, IL-17A expression is increased in the spleen of Lohmann and ISA Brown chickens intravenously infected with Salmonella Enteritidis (Matulova et al., 2012). Expression levels of the IL-17A and IL-17F are known to be elevated on day 4 and 7 in the spleen and liver of chickens infected with Salmonella gallinarum, but not in chickens infected with S. Typhimurium, which display an increased IL-17A and IL-17F expression in the spleen on day 7 post-infection (Kim et al., 2014). Interestingly, IL-17A knockout mice challenged with S. Enteritidis display increased bacterial burden in the spleen and liver compared with uninfected mice (Schulz et al., 2008). Salmonella spp. colonize most specifically the ceca and cecal tonsils (Crhanova et al., 2011) and S. Typhimurium infection induces proinflammatory cytokines IL-1 and IL-6 in the intestinal tissues of chickens (Fasina et al., 2008). We therefore monitored duIL-17A and duIL-17F transcript levels in the cecal tonsil of Salmonella-infected ducks. Expression levels of duIL-17A and duIL-17F transcripts were statistically unchanged in the cecal tonsils (Fig. 4C) and cecum (data not shown) in S. Typhimuriuminfected ducks as compared to the uninfected ducks. It
is interesting to note that IL-17A mRNA expression is unchanged in the cecum of chickens orally infected with S. Enteritidis (Matulova et al., 2012), and significantly downregulated in the ileum mucosa of pigs orally infected with S. Typhimurium (Collado-Romero et al., 2012). By contrast, increased expression of IL-17A transcript is detected in the cecum of chickens orally infected with S. Enteritidis (Crhanova et al., 2011), and in the ceca of streptomycinpretreated mice orally infected with S. Typhimurium (Godinez et al., 2008). Generally, expression levels of duIL17A and duIL-17F mRNA in Salmonella-infected ducks were weakly increased compared to Salmonella-infected chickens (Kim et al., 2014). We therefore monitored S. Typhimurium infection in the spleen and liver postinfection, and were able to identify bacteria in both organs in infected ducks (Fig. 4D). Although Salmonella infections in ducks and chickens are probably the most important source of Salmonellaassociated foodborne diseases in human, basic information about Salmonella control in duck industry depends mainly on research on salmonellosis in chickens, which may not be directly transferable to ducks. We cloned duIL17F and investigated expression patterns and functions. In Salmonella-infected ducks duIL-17A and duIL-17F levels were unchanged or weakly elevated in several tissues tested in contrast to Salmonella-infected chickens. Taken together, these findings suggest that mRNA expression levels of IL-17A and IL-17F in response to Salmonella infection
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