Dynamic Expression Pattern of SERPINA1 Gene from Duck (Anas platyrhynchos)

SERPINA1 is a member of serine protease inhibitors and is increasingly considered to be a regulator of innate immunity in human and animals. However, the expression and function of SERPINA1 gene in immune defense against viral infection remain unknown in ducks. The full-length du SERPINA1 cDNA sequence was obtained using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). It contained 1457 nucleotide, including 47-bp 5' UTR, 135-bp 3' UTR, and 1275-bp open reading frame (ORF), and encodes a 424-amino acid protein. Then, the tissue expression profile of du SERPINA1 gene was determined. Real-time quantitative polymerase chain reaction (real-time qPCR) analysis revealed that du SERPINA1 mRNA is ubiquitous in various tissues, but higher expression levels were observed in lung and liver tissues. In addition, the expression pattern was investigated when the ducklings were challenged with duck hepatitis virus 1(DHV-1) and polyriboinosinic polyribocytidylic acid (poly I:C). After DHV-1 injection or poly I:C treatment, du SERPINA1 mRNA was up-regulated in the liver and kidney tissues. However, the peak time in two tissues was not consistent. In kidney, the expression lever of SERPINA1 increased immediately after the treatment while in liver tissue it kept steady until 12 h post-infection. Our results indicate that SERPINA1 has an active role in the antiviral response, and thus improve our understanding of the role of this protein.


Introduction
Serpins, as a superfamily of serine protease inhibitors, play a vital role in complement regulation inflammation, angiogenesis, tumor suppression, apoptosis and other physiological processes [1,2]. There is ample clinical evidence that mutation in this gene could cause emphysema or liver disease, which showed a serious impact on the function and homeostasis of tissues and organs [3]. So far, sixteen clades have been identified, designated A through P, with an additional 10 serpins that are unclassified "orphans" [1]. SERPINA1, also known as Alpha-1 anti-trypsin (AAT), is a vital member of the SERPIN superfamily, which plays an important role in anti-inflammatory properties [4][5][6]. Recent findings indicate that Alpha1-antitrypsin may not only prevent damage from proteolysis but may also specifically degrade elastin in tissues and organs, and inhibit some immune pathways to affect regulation of innate immunity [4][5][6].
Ducks, as a feasible model, play an important role in studying avian influenza and human hepatitis, and has raised interest in the duck immune system [7]. DHV-1 is a small RNA virus causing high mortality in ducks (Anas platyrhynchos), especially in younger ducklings. In order to reduce the effect of DHV-1, a large number of studies have been reported on this virus [8][9][10][11][12][13][14]. In our previous study, we identified differentially expressed sequence tags (ESTs) of SERPINA1 using a suppression subtractive hybridization (SSH) cDNA library of 3-day-old ducklings treated with DHV-1 [15]. Our study showed that during DHV-1 and poly I:C infection, the expression of SERPINA1 mRNA was upregulated. In this study, we aim to expand those preliminary results, by assessing tissue-specific gene expression and the dynamic expression change of the SERPINA1 gene against the virus and thereby provide a theoretical basis for future immune pathological studies. . We also confirm that all efforts were made to minimize suffering.

Ducks, Challenge Experiments, and Sample Collection.
The 120 three-day-old domestic ducklings (Jingding duck) were purchased from the Chinese Waterfowl Germplasm Resource Pool (Taizhou, China). RT-PCR was used to make sure that the ducklings had not been exposed to DHV previous to our study [16]. Then, the ducklings were randomly divided into three groups, the 40 ducklings were injected with 0.4 mL of DHV-1(ELD50 10 −4.6 /0.2ml) according to our earlier trials [17,18], and the 40 ducklings were injected with 0.4 mL of poly I: C (0.5 mg/mL, Invivogen, California, USA), another 40 treated with normal saline (as uninfected controls). Injection dose and injection method are consistent with our earlier trials [18]. Besides, the ducklings after the infection showed the typical symptoms of hepatitis by carrying the DHV-1 virus, and the relative results have been published [17]. Additionally, five healthy three-day-old domestic ducks (Jingding duck, Anas platyrhynchos) were purchased from the Chinese Waterfowl Germplasm Resource Pool (Taizhou, China). Tissues, including liver, spleen, lung, heart, kidney, thymus, breast muscle and leg muscle, were obtained after euthanasia that the ducklings were immediately anesthetized with sodium pentobarbital (intraperitoneal injection; 150 mg/kg) and killed by exsanguination. These birds were referred to as morbid ducklings. The tissues were snap-frozen in liquid nitrogen immediately and stored at -80 ∘ C.

RNA Extraction and Cloning of du SERPINA1 cDNA.
Total RNA was isolated from the liver of ducks with TRIzol (Invitrogen) according to the manufacturer's instructions, and the quality of the isolated RNA was assessed by visualizing the ribosomal RNA bands after electrophoresis on a 1.0% agarose gel. The PrimeScript6 1st Strand cDNA Synthesis Kit (TAKARA, Dalian, China) was used according to the manufacturer's instructions with 1 g of total RNA as a template to produce cDNA. Then the polymerase chain reaction (PCR) amplification was conducted using LA Taq (TAKARA) with the following conditions: 94 ∘ C for 5 min, 35 cycles of 94 ∘ C for 30 s, 50 ∘ C for 30 s, and 72 ∘ C for 1 min 30s, followed by one cycle of 72 ∘ C for 10 min. Rapid amplification of cDNA ends (RACE) was used to obtain the 5' and 3' ends of du SERPINA1 using the SMART RACE cDNA amplification protocol (Clontech, Mountain View, CA, USA) and the 3'-Full RACE Kit (TaKaRa, Dalian, China), respectively. Gene-specific primers used for the amplification of RACE cDNA fragments were designed based on the obtained SERPINA1 nucleotide sequence. The sequences of du SERPINA1 was submitted to GenBank under the accession number KY471047. All the primer sequences mentioned above are shown in Table 1.

Bioinformatics
Analysis. Bioinformatic analysis of du SERPINA1 was performed using a software program like DNASTAR, the NCBI website (http://www.ncbi.nlm.nih.gov) and BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignment was created by AlignIR V2.0 and CLC Sequence Viewer 6 was performed to construct multiple sequence alignments of the amino acid sequences of du SERPINA1 proteins. The neighbor-joining phylogenetic tree was constructed based on the alignment result using the Neighbour-Joining (NJ) algorithm within the MEGA 6.0 program.

Quantitative Real-Time Polymerase Chain Reaction.
Total RNA was extracted from different tissues using TRIzol (Invitrogen), and 1 g total RNA was used with FastQuant RT Kit (With gDNase) (Tiangen, Beijing, China) during reverse transcription. The process included an initial phase at 42 ∘ C for 3 min, then incubation at 42 ∘ C 15 min, and incubation at 95 ∘ C for 3 min. The cDNA was stored at -80 ∘ C. Realtime qPCR was carried out on Applied Biosystems 7500 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA) with the following program: 1 cycle at 95 ∘ C for 5 min, followed by 40 cycles of 94 ∘ C for 30 sec, 60 ∘ C for 30 sec, and 72 ∘ C for 30 sec, and a final incubation at 72 ∘ C for 10 min. Relative quantitative of gene expression was calculated using the 2 −ΔΔCt method [19]. The GAPDH gene was used as an internal standard for relative expression levels. In addition, at each time point, the mean ΔCt value of control ducks was used to calibrate analysis of expression patterns in infected ducks. Therefore the results calibrated by the expressions of control ducks are displayed in the plot of du SERPINA1 expression of liver and kidney after DHV-1 and poly I: C challenge. All the primers are listed in Table 1.

Enzyme-Linked Immunosorbent Assay (ELISA) Analysis.
For ELISA analysis, by using double antibody sandwich method to determine the levels of IFN-, IFN-, IgG and IL-8, duck blood were gathered, and supernatants were collected after centrifugation at 3000 rpm for 20 min at 4 ∘ C and subjected to ELISA for detection of duck IFN-, IFN-, IgG and IL8. The concentrations of IFN-, IFN-, IgG and IL8 in the samples were measured with a multifunctional microplate reader (Tecan Infinite M200 PRO; Switzerland) and determined by comparing the optical density of the samples to the standard curve.

Statistical
Analysis. Data were analyzed using SPSS 22.0, and differences among tissue samples were assessed with oneway ANOVA. P values less than 0.05 were considered as significant. All data were processed by GraphPad Prism 5.0. Comparing of the full-length aa sequence of du SER-PINA1 to the SERPINA1 gene of other species by CLC Sequence Viewer 6, multiple alignments and amino acid sequence are shown in Figure 1(a). The duck SERPINA1 shared high similarity with SERPINA1 proteins from other vertebrates. Compared to chickens protein from the GenBank database, the du SERPINA1 protein had identities of 99 %. A condensed phylogenetic tree was constructed based on the derived amino acid sequences of SERPINA1 of organisms (Figure 1(b)). The overall tree topology revealed the following three major groups: bird, mammal and fish.

Tissue-Specific Expression Profile of du SERPINA1 Gene.
Real-time qPCR was carried out to determine the tissuespecific expression profile of du SERPINA1 gene in healthy ducks. The results showed that du SERPINA1 mRNA was widely expressed in all 8 tissues tested (liver, spleen, lung, heart, kidney, thymus, breast muscle and leg muscle). The expression levels of du SERPINA1 mRNA were more significant higher in lung and liver tissues, whereas the expression level in other tissues was extremely low (Figure 2).

The Expression of Cytokine following DHV-1 and
Poly I:C Treatment. In order to investigate the changes of cellular immunity and humoral immunity in ducklings during DHV-1 and poly I:C infection, the ELISA analysis was used to detect the changes of serum IgG, IFN-and other cytokines. The results showed that the cytokines, including IFNand IFN-generally indicated the fluctuating trend, which performed the trend of first increasing, then decreased, and then increased (Figures 3(a) and 3(b)), while IL8 and IgG did not change much as a whole (Figures 3(c) and 3(d)).

du SERPINA1 Expression after DHV-1 and Poly I:C
Challenge. To further define the expression change of SER-PINA1 during DHV-1 or poly I: C treatment, time-dependent expression levels in the liver and kidney after DHV-1 or poly I: C challenge were characterized (Figure 4). In the liver upon DHV-1 treatment, expression of du SERPINA1 increased at 12 h post-infection (h.p.i), and reached peak at 48 h.p.i, after this peak, expression of du SERPINA1 then progressively dropped. In the poly I: C treatment group, expression of du SERPINA1 increased at 36 h.p.i, reaching a peak at 72 h.p.i, and then decreased dramatically (Figure 4(a)). The expression of du SERPINA1 in kidney was increased immediately at 4 h.p.i with DHV-1 and poly I: C (Figure 4(b)).

Discussion
SERPINA1, also known as Alpha-1 anti-trypsin (AAT), is a multifunctional protein with proteinase inhibitory, antiinflammatory and cytoprotective properties. Some studies have shown AAT could prevent the lethality of TNF or endotoxin in mice [20]. It is also illustrated in human that AAT can decrease the release of TNF-from LPS stimulated and unstimulated monocytes in vitro. Besides, in most circulation, AAT was synthesized in the liver, and when suffering an acute phase of inflammation or infection, the AAT was released rapidly [21].
In healthy ducks, the results of tissue-specific transcriptional profile of du SERPINA1 showed the uneven expression in different tissues.   : du SERPINA1 mRNA expression in various tissues (liver, spleen, lung, heart, kidney, thymus, breast muscle and leg muscle). All assays were repeated at least three times, and data are shown as mean ± S.E. (n = 5) from one representative experiment. The expression of SERPINA1 was normalized to GAPDH. Different letters showed significant difference (p < 0.05). liver was significantly higher than in other tissues, supporting the fact that SERPINA1 was attributed to different roles in physiology, particularly including immune responses associated with liver disease, which was similar with the expression pattern of SERPINA1 gene in human and mice [22,23]. Besides, the high expression of SERPINA1 was also existed in lung, which was consistent with previous studies that SERPINA1 may attenuate inflammation in ventilatorinduced lung injury by modulating inflammation-related protein expression [24,25].
Cytokines can regulate the physiological functions of leukocytes, mediate inflammatory responses, participate in immune responses, and repair tissue. In order to further study the changes of host immune and inflammatory response caused by DHV-1 and poly I: C infection, the levels of cytokines in serum were tested during the process of infection. IFN-, IFN-, as immunomodulatory pleiotropic factors, have been used as an indicator of cell-mediated immunity in infected organisms [26], and IL8 acts as an inflammatory cytokine and participates in the host's inflammatory response. In this study, IFN-and IFN-got peaked at the early stage after DHV-1 and poly I:C infection, and the changes of IL8 and IgG were not significant after infection with DHV-1 and poly I:C [27,28]. These results revealed the cells of the immune system was directly damage after viral infection and T-cell immunity played a major role in the process of DHV-1 and poly I:C infection, supplementing by B-cell immunity. Subsequently, in order to explore the dynamic expression pattern of du SERPINA1 in ducks during viral infection, the du SERPINA1 mRNA expression after DHV-1 or poly I: C challenge was detected. Previous studies have showed that DHV-1 rather than other DHV types could specifically infect duck embryo liver and duck embryo kidney cells [29,30] and in this study, liver and kidney tissues were selected to be candidate tissues. During the early period of viral injection, the expression of SERPINA1 was no significant difference in the liver, while it was rapidly increased in the kidney tissue, which may be related that during suffering the infection of the viral antigen, the host's various non-specific defenses, such as natural killer lymphocyte cell immunity, secrets more IFNand IFN- [31] to regulate the expression of SERPINA1 gene regulation pathway, which lead to an increase in early SERPINA1 gene expression. After achieving the peak, the expression of du SERPINA1 decreased to a normal level, which may be prevented excessive inflammatory reaction. In pig [32], when the expression of du SERPINA1 was upregulated after PCV2 infection, the immune factor was transported to the immune related tissues through the circulation of blood, which prevented excessive inflammatory reaction and maintained the integrity and normal function of the immune tissue. With the increase of age, the immune system of ducklings is becoming more and more adaptive. Studies have shown that the immune level of 7-day-old chicken is comparable to that of adult chickens [6], and the mature immune system will produce a large number of cytokines to resist the attack of virus. These results suggested SERPINA1 might be associated with the host defence response against viral infection. The specific mechanism is unclear but may autocrine regulation of SERPINA1 mRNA synthesis when virus invasion.
Previous studies have proved that SERPINA1 has a vital role in protect host tissue against injuring at sites of inflammation. Now, increasing evidence showed that SERPINA1 (AAT) may exhibit biological activity independent of its protease inhibitor function [33,34]. From our results, we speculated that when being attacked by virus, the host needs more SERPINA1 which is consistent with the previous research results. To conclude, our results provide a better understanding of du SERPINA1 function in immunity during viral infection.

Conclusions
In this study, we first cloned and characterized the gene in duck and demonstrated that the du SERPINA1 gene shared high similarity with SERPINA1 proteins from other vertebrates. Transcriptional analyses showed ubiquitous expression of SERPINA1 gene in eight examined tissues. Expression analyses showed that SERPINA1 gene was significantly upregulated in vivo after DHV-1 or poly I: C stimulation. Taken together, our results provide a better understanding the dynamic expression change of SERPINA1 gene against virus and thereby provide a theoretical basis for future immune pathological studies.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.