Developmental and Tissue Patterns of the Basal Expression of Chicken Avian β-Defensins

Defensins are a class of antimicrobial peptides in vertebrates that function as the first line of innate immunity with potent antimicrobial and immunomodulatory activities. Fourteen defensins, namely, avian β-defensin 1 to 14 (AvBD1-14), have been identified in chickens. Before characterizing the role of AvBDs in innate immunity during the early development of chickens, we collected tissue segments from the liver, spleen, and gastrointestinal (GI) tract including the esophagus, crop, proventriculus, gizzard, duodenum, jejunum, ileum, cecum, and colon from broilers at days 1, 3, 7, 14, and 28. After RNA isolation and reverse transcription, we determined the expression levels of the 14 AvBD genes in these tissues during the first 28 days after hatching by real-time PCR. The results suggested the AvBDs were widely expressed in the chicken liver, spleen, and gastrointestinal (GI) tract. Interestingly, we did not detect AvBD11 expressed in the GI tract, even in the liver and spleen. Additionally, AvBDs were differentially expressed in the chicken GI tract. AvBD5 and AvBD14 were expressed most abundantly in the proximal GI tract, especially the esophagus and crop. Moreover, AvBD5, AvBD7, AvBD9, and AvBD14 were expressed in an inverted-V pattern with the peak being the observed expression at days 3, 7, or 14 in the chicken spleen, esophagus, duodenum, and cecum. Other AvBDs presented biphasic or inverted-V expression patterns in different tissues. The expression levels of all detected AvBDs were strengthened after hatching rather than decreasing steadily. Therefore, AvBDs were found to be expressed widely in the chicken liver, spleen, and GI tract and their expression levels were primarily up regulated during the early development of chicken, implying the potential essential roles of AvBDs in early innate defense and infection resistance of chickens.


Introduction
Due to the overuse of antibiotics in farm animals, it would raise a high risk of transferring antibiotic resistance to humans, which may threaten public health. Therefore, the development of novel antibiotics is an urgent need and is attracting increasing attention. Enhancing the synthesis of host defense peptides (HDPs) has emerged as a hostdirected antibiotic alternative therapy showing less likelihood of triggering antibiotic resistance [1,2]. As an essential part of innate immunity, HDPs, also known as antimicrobial peptides, are small peptides consisting of less than 100 amino acid residues. These peptides include various groups of small peptides defending against environmental pathogens.
For vertebrates, HDPs consist of two major families: cathelicidins and defensins. HDPs are mainly synthesized and secreted by phagocytic cells and the cells of the epithelial surfaces such as the skin and the respiratory, gastrointestinal (GI), urogenital, and reproductive tracts [3,6], suggesting that HDPs act as the first line of defense against microbes. Chickens express 4 cathelicidins (CATH) and 14 avian βdefensins (AvBDs) with no αand θ-defensins, namely, CATH1-3 (also known as fowlicidin 1-3), CATH-B1, and AvBD1-14 [7]. Accumulating evidence indicates that AvBDs have wide-spectrum microbiostatic activities against gramnegative and gram-positive bacteria, fungi, and viruses [8,9]. For instance, purified recombinant AvBD6 protein could inhibit the growth of Escherichia coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Saccharomyces cerevisiae, and Candida albicans [10]. Recombinant AvBD2 was able to lower the cytotoxicity of the Newcastle disease virus [8]. Exhibiting potent antimicrobial activities, HDPs are considered a potential antibiotic alternative strategy.
The GI tract acts as the digestive tract to extract nutrients from food for chickens and includes the esophagus, crop, proventriculus, gizzard, duodenum, jejunum, ileum, cecum, and colon [11]. AvBDs synthesized by epithelial cells lining the GI tract are essential weapons by which chickens could combat the millions of microbes trying to invade through different pathways [9,12,13]. In the GI tract, AvBD9 is one of the most widely spread AvBDs and is found in the esophagus, crop, proventriculus, gizzard, duodenum, ileum, and colon [10,14,15]. However, few studies have been conducted on the developmental and tissue patterns of AvBD basal expression in chickens at an early age.
In this study, we examined the developmental and tissue expression patterns of AvBDs in broiler chickens during the first 28 days after hatching. We noticed that each AvBD had tissue specificity and that AvBDs would give a higher expression during the first two weeks than on day 1 after hatching, indicating that AvBDs are significant for chickens to fight against pathogens during early development.

Materials and Methods
2.1. Animals and Sampling. The experiments were approved by the Animal Care and Use Committee of Zhejiang Academy of Agricultural Sciences. A total of 90 newly hatched male Ross 308 broiler chicks were raised in a controlled environment at a temperature of 34-35°C in the first week followed by a reduction of 3°C weekly to a final temperature of 26°C. The broilers received no antibiotics throughout the experimental period. Tissue samples (n = 9) were collected on days 1, 3, 7, 14, and 28 from the esophagus, crop, gizzard,  [17]. Ori-ginPro 2018 (OriginLab Corporation, Northampton, MA, USA) was used for data visualization and analysis with oneway ANOVA. The data are expressed as means ± standard error of means (SEM). P value < 0.05 was considered to indicate significant differences.

Abundance of AvBDs in Chickens.
To evaluate the expression abundance of AvBDs in the chicken GI tract, we collected various tissues along the chicken GI tract from eighteen 14-day-old Ross broiler chickens and subjected the tissues to total RNA isolation and RT-qPCR. The average ΔCt values were calculated to indicate the AvBD1-14 expression abundances, with normalization to GAPDH expression. As expected, all AvBDs except AvBD11 were widely expressed in the chicken esophagus, crop, proventriculus, gizzard, duodenum, jejunum, ileum, cecum, and colon with different levels of abundance ( Figure 1). The small SEM implies that each AvBD gave a similar expression level in each segment of the chicken GI tract. As shown in Figure 1, among all the detected AvBDs, AvBD1 was the most abundantly expressed (mean of ΔCt = 6:70) while AvBD13 was the least abundant one (mean of ΔCt = 14:37) in the chicken GI tract. Additionally, AvBD11 was expressed at the lowest level in the chicken GI tract, as its expression was not detected in the present study.

Differential Tissue Pattern of Chicken AvBDs.
To study the tissue expression patterns of AvBDs along the chicken GI tract, we collected tissue samples from the liver, spleen, and the GI tract, including the esophagus, crop, proventriculus, gizzard, duodenum, jejunum, cecum, and colon, from 7- ΔCt value 8 day-old Ross chickens because most of the AvBD expression was expressed at relatively high levels in 7-day-old chickens.The basal mRNA expression levels of AvBD1-14 were examined by RT-qPCR after RNA extraction and reverse transcription. The average ΔCt values of all the detected AvBDs in the chicken GI tract ranged from 6.70 to 14.37, and the average was 10.87 ( Figure 1). Accordingly, among all the detected defensins, all but AvBD11 were widely expressed with different patterns along the chicken GI tract and in the liver and spleen ( Figure 2). As shown in Figure 2, AvBDs 1-4, 6, and 7 shared a common expression pattern; i.e., the chicken spleen exhibited the highest expression levels of those β-defensins compared to other tissues at 7-day-old, whereas the duodenum gave the highest expression level in AvBDs 1-4, 6, and 7. Additionally, the difference between the highest and lowest relative mRNA expression levels of AvBDs 1-4, 6, and 7 was more than 40-fold and was as high as 178-fold ( Figure 2). Another differentially expressed β-defensin was AvBD9; the difference between the highest and lowest expression was as much as 25-fold. However, AvBD9 was expressed at a relatively higher level in the proventriculus than in other tissues, even compared to the levels in the liver and spleen. Besides, the liver showed a higher AvBD9 mRNA expression level than the spleen. For the other AvBDs (AvBD5, AvBD8, AvBD10, AvBD12, and AvBD13), the mRNA expression levels in different tissues were not very different ( Figure 2). The relative fold changes of these AvBDs were no more than 15-fold. AvBD11 was not detected in the chicken GI tract because the absolute Ct value of AvBD11 was above 35.    Figure 2: Tissue patterns of chicken AvBDs along the GI tract, liver, and spleen. Tissue segments of the esophagus, crop, proventriculus, gizzard, duodenum, ileum, cecum, colon, liver, and spleen were obtained from chickens at 7-day-old. After RNA isolation, RT-qPCR analysis was performed to evaluate the mRNA expression levels of all chicken AvBDs. Fold changes of chicken AvBD expression were calculated relative to the expression level of the colon on day 28 using GAPDH as the reference gene and are expressed as the mean ± standard error of the mean of nine chickens.

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Among chicken digestive tissues, it was also observed that the duodenum yielded the highest expression of AvBDs 1-4, 6, and 7, while the crop and proventriculus gave the highest expression levels of AvBD5, AvBD13, AvBD8, and AvBD9, respectively ( Figure 2). Furthermore, AvBD12 and AvBD14 were highly expressed in the gizzard and esophagus, respectively. Esophagus and crop shared the same patterns of the AvBD expression (Supplemental Figure (see available here)). Particularly, AvBD10 was expressed most in the colon, duodenum, and cecum with up to 7.14-fold change while AvBD9 was the most abundantly expressed in the proventriculus with fold change up to 24.82 (Supplemental Figure (see available here)).

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International observed on day 14 or 28 relative to day 4 ( Figure 3). The mRNA expression levels of AvBD2, AvBD4, AvBD6, and AvBD10 showed similar patterns, with the expression peak on day 14 ( Figure 3). Interestingly, the highest expression of AvBD8 was observed on day 28, where the difference was significant (P < 0:001). Significant downregulations (P < 0:01) in the expression levels of AvBD1, AvBD3, AvBD5, AvBD7, AvBD9, AvBD12, and AvBD14 were observed in the spleen on day 28 relative to day 3 while the expression levels of AvBD2, AvBD6, and AvBD10 significantly attenuated on day 28 relative to day 14 (P < 0:001). The mRNA expression level of AvBD1 was significantly decreased by nearly 14-fold between day 3 and day 28 (Figure 3).
In the digestive tract, the AvBD expression patterns were more complex. Biphasic expression patterns showed up in the esophagus (Figure 4), duodenum ( Figure 5), cecum (Figure 6), and other digestive tissues (Supplemental Figure (see available here)). In the esophagus, AvBD1, AvBD4, AvBD6, AvBD8, AvBD9, AvBD12, and AvBD13 gave a biphasic expression pattern, where these genes were abundantly expressed on day 1 or day 3 but eventually reduced to the lowest expression on day 7 or day 14 ( Figure 4). Additionally, AvBD2, AvBD3, AvBD5, AvBD7, AvBD10, and AvBD14 showed peak expression levels at different days with the lowest expression being observed on day 28. The duodenum and cecum showed similar expression patterns (Figures 5 and 6). Interestingly, no : Developmental expression of chicken β-defensins in the esophagus. Esophagus segments were harvested from broilers at 1, 3, 7, 14, and 28 days of age and were subjected to RT-qPCR analysis after RNA isolation and reverse transcription. The mRNA expression levels of βdefensins at the indicated ages were calculated as fold changes relative to the expression level on day 28 using GAPDH as the housekeeping gene. Each bar represents the mean ± standard error of the mean of three chickens. The difference was considered significant using one-way ANOVA followed by Tukey's test. * P < 0:05; * * P < 0:01; * * * P < 0:001.

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BioMed Research International significant difference in AvBD14 expression level was observed in the cecum at different ages ( Figure 6). For AvBD8, the spleen, esophagus, and duodenum shared the same expression pattern, where AvBD8 expression was increased to the first peak on day 3 but slightly reduced from day 7 to day 14, followed by the highest expression on day 28.

Correlations among Chicken AvBD Expression in the Chicken Digestive Tract. To reveal the correlations among
AvBDs expression in the chicken GI tract, correlations were determined using the Pearson correlation coefficient. The expression levels of half of the AvBDs were significantly correlated with chicken age, suggestive of the age specificity of AvBD expression. As stated in Table 2, the correlation coefficients of AvBD2 vs. AvBD4, AvBD4 vs. AvBD6, AvBD4 vs. AvBD7, and AvBD6 vs. AvBD7 were all more than 0.8 with high significance (P < 0:01), showing a strong positive correlation between these AvBD expressions.

Discussion
The digestive system in chickens consists of the esophagus, crop, proventriculus, gizzard, small intestine (duodenum, jejunum, and ileum), cecum, colon, and cloaca [18]. The broad AvBD distribution along the digestive tract makes these organs function as barriers in preventing pathogen invasion into the GI tract [7]. The present study showed  The duodenums were harvested from broilers at 1, 3, 7, 14, and 28 days of age and were subjected to RT-qPCR analysis after RNA isolation and reverse transcription. The mRNA expression levels of βdefensins at the indicated ages were calculated as fold changes relative to the expression level on day 28 using GAPDH as the housekeeping gene. Each bar represents the mean ± standard error of the mean of three chickens. The difference was considered significant using one-way ANOVA followed by Tukey's test. * P < 0:05; * * P < 0:01; * * * P < 0:001.

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AvBDs were widely expressed in the chicken GI tract except for AvBD11, which coincides with the previous study [9,15]. Although the expression levels of AvBDs and other host defense peptides gave a gradual increase followed by a dramatic decrease in the sterile environment during the embryonic development of chickens [19,20], we found the AvBD expression levels were further strengthened during the first 28 days after hatching. Because chicks are constantly exposed to different pathogens in the ambient environment with inadequate protection of circulating maternal antibodies [21], these results might not be surprising. With the presence and rapid development of innate immunity in chicks, the adaptive immunity gradually matures to prevent various pathogen infections in newly hatched chickens. The heightened expression of AvBDs could contribute essential protection against environmental microbial invasion in the early stage of chickens with potent antimicrobial activities. For detected AvBDs, different tissues showed their preferences for AvBD expression. For instance, AvBD1-4 and AvBD6-7 were strongly expressed in the duodenum, and AvBD5 and AvBD13-14 were strongly expressed in the proximal GI tract including the esophagus and crop (Figure 2 and Supplemental Figure (see available here)). Additionally, the high expression levels of AvBD8-9 and AvBD12 were observed in the proventriculus and gizzard, respectively. Interestingly, the distal GI tract was relatively rich in only AvBD10 and AvBD12. AvBDs function not only as antimicrobials but also as sensors of the host-microbiome balance along the digestive tract [22]. Our results provide evidence that the cecum might be tolerant to microbes for further feed digestion. We also found similar patterns of AvBD expression in the esophagus and crop, possibly because the only  Figure 6: Developmental expression of chicken β-defensins in the cecum. The ceca were harvested from broilers at 1, 3, 7, 14, and 28 days of age and were subjected to RT-qPCR analysis after RNA isolation and reverse transcription. The mRNA expression levels of β-defensins at the indicated ages were calculated as fold changes relative to the expression level on day 28 using GAPDH as the housekeeping gene. Each bar represents the mean ± standard error of the mean of three chickens. The difference was considered significant using one-way ANOVA followed by Tukey's test. * P < 0:05; * * P < 0:01; * * * P < 0:001.  9 BioMed Research International mucosal glands in the crop are located in the junction of the esophagus and crop [11].

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The majority of defensins are broadly expressed in a wide range of cells in the GI tract [22,23]. Typically, some α-defensins are uniquely synthesized in Paneth cells in the crypts in the small intestine [24][25][26]. However, the chicken genome has no α-defensin gene, and a recent study indicated the presence of Paneth cells in the chicken small intestine [27]. These observations make it particularly interesting to investigate the expression patterns of βdefensins in the chicken GI tract. Our results showed that all but AvBD11 were widely expressed in the tissues of the GI tract. The absence of AvBD11 mRNA in the GI tract is probably due to its unique gene structure, which contains two tandem copies of the six-cysteine motif in the mature peptide [28]. However, the expression of AvBD11 was detected in the crop and intestinal mucosal layer of Cobb and Ross broiler chickens and its expression would increase in the intestinal mucosal layer when the Cobb broilers were challenged with Eimeria maxima [29]. The chicken species used in this study and our focus on the basal expression level of AvBDs might be another reason why we did not detect AvBD11 expression. Simultaneously, we noticed that multiple significant correlations were present among some genes. Coupled with the locations and phylogenetic relationships of chicken defensins, we also found that many of the genes expressed with high correlations are located close or even adjacent to each other and are clustered in the same gene clade. For instance, all of the correlations among AvBD1-4 were significant with correlation coefficient values higher than 0.5 ( Table 2). Regarding AvBD6, which is a duplicate from AvBD7 in Galliformes with a short chromosome distance [15], it also presented a strong positive correlation with AvBD7. Interestingly, significant correlations with high correlation coefficients are also observed in some genes with long chromosome distances and low similarities. For example, the correlation between AvBD9 and AvBD13 is highly significant with a correlation coefficient of 0.88, while the two genes not only show less similarity at the amino acid level but are also located in different AvBD gene blocks.
As represented by AvBD1, the expression levels of multiple AvBDs tended to be decreased from day 1 to day 28 in both the GI tract and the liver and spleen. The decreased expression levels of AvBDs might be associated with the maturation of adaptive immunity. In contrast, the mRNA abundance of AvBD8 pervasively increased along with age, which is notably different from the other AvBDs. On the account of the narrow antibacterial range of AvBD8 in chickens, the primary biological function of this gene is unlikely to be antimicrobial [30]. In fact, defensins represent a posse of pleiotropic molecules that exert many other effects in the immune system beyond host defense. Therefore, the gradual increasing of the AvBD8 expression in these tissues may relate to other functions or may act synergistically with other defensins to exert defense functions. Different from the liver and spleen, the GI tract continuously interacts with microbes. It has been known that defensins are able to balance among bacterial populations and to control homeostasis in the GI tract [31]. Chicken CHCC-OU2 cells challenged in vitro by commensal gut bacteria lead to significant expression changes in some AvBDs [32]. Therefore, some biphasic expression patterns, such as the abrupt elevation in the duodenum at day 7, may be the result of microbe colonization. Meanwhile, some microbial productions can also stimulate the expression of chicken AvBDs. Butyrate, a kind of short-chain fatty acid produced by Clostridium butyricum, is capable of inducing a group of AvBDs in multiple cell types [33]. This finding may indirectly regulate the expression of some AvBD genes in the GI tract. The expression levels of AvBDs in the liver and spleen are abundant because they are important immune organs with a large amount of immune cells. The different expression patterns of AvBDs in these tissues may also be associated with their biological functions.

Conclusions
Taken together, the expressions of AvBDs in the GI tract are gene-, tissue-and age-specific. Influenced by the living environment, health condition, and genetic background, conflicting expression patterns are observed for AvBDs in different studies [9,28,32]. Continued studies focus on the identification of the cell types that synthesize specific AvBDs and investigations of AvBD mRNA abundance by using germ-free chickens, which would improve our understanding of the expression patterns of this gene family.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare no conflict of interest.