Tissue expression and developmental regulation of chicken cathelicidin antimicrobial peptides
© Achanta et al.; licensee BioMed Central Ltd. 2012
Received: 26 April 2012
Accepted: 9 May 2012
Published: 31 May 2012
Cathelicidins are a major family of antimicrobial peptides present in vertebrate animals with potent microbicidal and immunomodulatory activities. Four cathelicidins, namely fowlicidins 1 to 3 and cathelicidin B1, have been identified in chickens. As a first step to understand their role in early innate host defense of chickens, we examined the tissue and developmental expression patterns of all four cathelicidins. Real-time PCR revealed an abundant expression of four cathelicidins throughout the gastrointestinal, respiratory, and urogenital tracts as well as in all primary and secondary immune organs of chickens. Fowlicidins 1 to 3 exhibited a similar tissue expression pattern with the highest expression in the bone marrow and lung, while cathelicidin B1 was synthesized most abundantly in the bursa of Fabricius. Additionally, a tissue-specific regulatory pattern was evident for all four cathelicidins during the first 28 days after hatching. The expression of fowlicidins 1 to 3 showed an age-dependent increase both in the cecal tonsil and lung, whereas all four cathelicidins were peaked in the bursa on day 4 after hatching, with a gradual decline by day 28. An abrupt augmentation in the expression of fowlicidins 1 to 3 was also observed in the cecum on day 28, while the highest expression of cathelicidin B1 was seen in both the lung and cecal tonsil on day 14. Collectively, the presence of cathelicidins in a broad range of tissues and their largely enhanced expression during development are suggestive of their potential important role in early host defense and disease resistance of chickens.
Antimicrobial peptides (AMPs) are an important component of the innate immune system playing a critical role in host defense and disease resistance in virtually all species of life [1–4]. AMPs generally consist of < 100 amino acid residues, mostly cationic and amphipathic in nature, which allows them to bind and disrupt negatively charged microbial membranes leading to cell death. Because of non-specific physical interactions with membranes, it is extremely difficult for bacteria to develop resistance. In addition to being antimicrobial, a number of AMPs were recently found to exert a broad range of immunomodulatory roles by recruiting and activating all major types of innate and adaptive immune cells [3, 5]. Therefore, AMPs are being actively developed for the control and prevention of infectious diseases, particularly against antibiotic-resistant bacteria .
Cathelicidins are a major family of AMPs in vertebrate animals including chickens. All cathelicidins are synthesized as prepro-peptides, with the prepro-sequence being highly conserved across species and the carboxyl terminal, biologically active mature sequence highly diversified . In mammals, besides the mucosal epithelial cells lining the digestive, respiratory, and reproductive tracts, cathelicidins are most abundantly expressed in myeloid progenitor cells and stored in neutrophil granules as pro-peptides, which are converted into active forms by proteolytic cleavage upon degranulation. The chicken genome was recently found to encode four genes for cathelicidins, namely fowlicidins 1 to 3 and cathelicidin B1 that span a 7.5-kb distance on chromosome 2 [7–10]. All four chicken cathelicidins are capable of killing a broad range of bacteria including antibiotic-resistant strains [9, 11–14]. Like in mammals, fowlicidin-2/CMAP27 was shown to be localized in the granules of chicken heterophils, equivalent to mammalian neutrophils, and processed into a mature form upon stimulation with bacterial lipopolysaccharide .
AMPs including cathelicidins have been detected in the meconium and feces of human infants , and elevated levels of cathelicidin have been noticed in the infants associated with respiratory infections , suggestive of the role of cathelicidins and other AMPs in early host defense of humans. In chickens, expression of fowlicidins 1 to 3 is detected as early as day 3 of embryonic development and then significantly increased as the embryo develops further, whereas cathelicidin B1 is not expressed until day 9, but significantly increased by day 12 in the developing embryo .
In this study, we studied the tissue expression pattern of four cathelicidins in 28-day-old broiler chickens and further examined their expression in the first 28 days after hatching. We observed that chicken cathelicidin transcripts are synthesized in a wide range of tissues and differentially expressed during the development, suggesting that cathelicidins may play an important role of early host defense of chickens.
Materials and methods
Tissue sampling and preparation
Day-old male and female Cornish Rock broiler chickens were purchased from a commercial hatchery (Ideal Poultry, Cameron, TX, USA) and reared under standard care in Laboratory Animal Resource Facility at Oklahoma State University, Stillwater, OK, USA. Tissues were collected from chickens of 2, 4, 7, 14, and 28 days, with 3 to 5 animals per age group. The range of tissues that were harvested included the crop, esophagus, proventriculus, gizzard, duodenum, jejunum, ileum, cecal tonsil, cecum, colon, lung, heart, trachea, liver, spleen, thymus, kidney, skin, breast muscle, brain, testis, ovary and bursa. All tissues were snap frozen in liquid nitrogen and stored at −80°C until used. Animal procedures were approved by the Institutional Animal Care and Use Committee of Oklahoma State University under protocol no. AG0610.
Isolation and quantification of total RNA
Tissues were homogenized in Tri Reagent (Sigma-Aldrich, St Louis, MO, USA), followed by total RNA extraction according to the manufacturer’s instructions. Air-dried RNA pellet was suspended in nuclease-free water and mixed thoroughly until the pellet was completely dissolved. RNA concentration and quality were measured using NanoDrop Spectrophotometer (NanoDrop Products, Wilmington, DE, USA).
Reverse transcription of total RNA
QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA) was used to synthesize the first-strand cDNA from total RNA following the manufacturer’s recommendations. Briefly, 0.3 μg of total RNA was first eliminated of genomic DNA contamination in a genomic DNA wipeout buffer for 5 min at 42°C. Reverse transcription was then performed in a total volume of 4 μL using Quantiscript reverse transcriptase and a mixture of random hexamers and oligo(dT) primers for 30 min at 42°C, followed by 3 min at 95°C to inactivate reverse transcriptase. The cDNA concentration was then measured using NanoDrop Spectrophotometer following a 10-fold dilution in nuclease-free water.
Real time PCR
Primer sequences of chicken cathelicidins for real time PCR
Forward primer (5' to 3')
Reverse primer (5' to 3')
Product size, bp
All data were analyzed with one-way ANOVA, followed by Tukey’s test using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). The results were considered significant, if P < 0.05.
Results and discussion
Tissue expression pattern of chicken cathelicidins
All four cathelicidin transcripts were found to be expressed in the testis and ovary (Figure 1). Additionally, heart showed an abundant expression of all four cathelicidins. It is noted that all primary and secondary lymphoid tissues including the bursa, thymus, spleen, and cecal tonsil express all four cathelicidins at moderate or high levels (Figure 1). In fact, bursa and bone marrow are the primary places for synthesis of cathelicidin B1 and fowlicidins 1 to 3, respectively (Figure 1) . Cathelicidin B1 was found earlier to be expressed in secretory epithelial cells surrounding M cells, a major portal of entry for pathogens in mucosal lymphoid tissues including bursa . It will be important to identify the cell types that express other cathelicidins in mucosal lymphoid tissues. At the same time, it will be interesting to reveal which cell type synthesizes cathelicidins in the spleen and thymus with no M cells present. Presence of cathelicidins in lymphoid tissues may be suggestive of a possible involvement of AMPs in the maturation and development of adaptive immunity. In fact, it is known that many AMPs including cathelicidins are capable of regulating adaptive immunity through activation of dendritic cells .
Expression of cathelicidins in a broad range of tissues including many non-immune tissue types also raised the possibility that these cathelicidins may play a role beyond host defense. Indeed, several AMPs have been shown to be involved in sperm maturation [19–21], consistent with the finding that a majority of defensins are most abundantly expressed in the male reproductive tracts in mammals .
As compared with mammalian species, chickens express a negligible amount of cathelicidins in the skin (Figure 1). This result could be attributed to the evolution of the skin in different species. The skin of birds is covered with feathers and many of the diseases in birds are contracted through oral and nasal routes. Hence, AMP synthesis in the skin might not be as much needed in birds than in mammals. In fact, a relatively high level of fowlicidin-2/CMAP27 expression was found in the uropygial gland , which secretes preen oil and antimicrobial factors that spread over plumage and provide protection against skin infections. On the other hand, as skin acts as an important route of entry for microorganisms in most mammals, it is not surprising to see a large amount of cathelicidins and other AMPs synthesized in the skin for protection.
Developmental expression of chicken cathelicidins
Overall, following a gradual increase in the expression of cathelicidins and many other innate host defense factors in a sterile environment during the embryonic development of chickens , we observed a further augmentation of cathelicidin expression during the first 28 days after hatching. The results are perhaps not surprising, given that newly hatched chickens are constantly exposed to various pathogens in ambient environments, albeit with the presence of circulating maternal antibodies, which are acquired through egg yolk, but are insufficient to provide adequate protection against microbial infections . In addition to maternal antibodies, the innate immune mechanisms must be present and develop quickly before the adaptive immunity matures. The availability and enhanced expression of cathelicidins may provide an important protection mechanism against the invading infections during the early stages of life in chickens. Consistently, chicken cathelicidins and other AMPs were shown recently to possess potent antimicrobial activities against a broad range of pathogens including many intestinal bacteria [9, 11–14].
However, it remains unclear in our study whether an enhanced expression of chicken cathelicidins during the early development is triggered by developmental signals or a consequence of constant exposure to microflora or environmental pathogens. To dissect it, chickens raised under germ-free conditions have been to be used. Nevertheless, many AMPs including a cathelicidin were detected in human meconium and neonatal fecal extracts, both of which indeed showed direct antibacterial activities . It is tempting to speculate that the presence of AMPs in the neonatal gut not only provides an important host defense mechanism, but also control initial colonization of intestinal flora. The amount and type of each AMP present in the gut may dictate the profiles of microbiota, given several different human AMPs exhibiting overlapping but not identical antimicrobial spectra .
In summary, all four cathelicidins are widely expressed in a broad range of chicken tissues, suggestive of their important innate defense role. Moreover, an augmented synthesis of the cathelicidin transcripts during the development coincides with the maturation of the immune system and a need for protection of the host in ambient environments. Our study of tissue and developmental expression of four chicken cathelicidins has shed new lights on the mechanisms of innate host defense and development of the immune system of chickens. Because of an association of single nucleotide polymorphisms (SNPs) in several chicken AMPs with animal resistance to Salmonella infection [26, 27], it is possible to genetically select chicken lines with enhanced disease resistance. Alternatively, dietary modulation of endogenous AMP expression represents another convenient approach to disease control and prevention in both humans and animals [28, 29].
This work was supported by a United States Department of Agriculture grant 2008-35204-04544, Oklahoma Center for the Advancement of Science and Technology grants HR07-113, and AR07.2-087, and Oklahoma Agricultural Experiment Station project H-2811.
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