Eighteen-day-old embryonic chicken eggs were used to culture the primary chicken TECs. The tracheae from the embryos were isolated, collected in 1× phosphate-buffered saline (PBS), and rinsed once with 1× PBS. The isolated tracheae were dissected into 1-cm sections after removing the surrounding muscle, connective tissue, and mucus. The trachea sections were incubated at 37°C for 2 h in culture medium containing collagenase type 1 (1 mg/mL; Gibco, Grand Island, NY, USA). To halt the enzymatic reaction, the tracheae were gently rinsed and dispersed in the culture media. The epithelial cell sheet was separated from the inner layer of the trachea and placed in a 15-mL tube. Cell pellets were obtained by centrifugation at 1,000×g for 5 min, and the cell pellet was resuspended in 0.1% trypsin-ethylenediaminetetraacetic acid for 5 min to dissociate into single cells. After preculture for 3 to 4 h in a cell culture dish (90×20 mm, #20100; SPL Life Sciences, Pocheon, Korea), the supernatant was collected and cultured by seeding 1.5 ×10⁶ cells in a 12-well plate (#13485; SPL Life Sciences, Korea). The collected cells were then cultured in a culture medium containing 1% L-glutamine (200 mM; Sigma-Aldrich, Saint Louis, MO, USA), 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, 1% penicillin/streptomycin in Dulbecco’s modified essential medium (DMEM)/F-12 (Gibco, USA), and 10% fetal bovine serum (FBS; Gibco, USA). Before use in cell cultures, the plates were coated with 0.1% gelatin at 37°C for 20 min.

The DF-1 chicken fibroblast cell line (ATCC CRL-12203, USA) was obtained from the American Tissue Culture Collection. DF-1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 Complete Medium (Thermo Fisher Scientific, Waltham, MA, USA) containing penicillin (100 IU/mL), streptomycin (100 mg/mL), and 10% FBS (Gibco, USA) in 5% CO₂ at 37°C in a humidified incubator. This cell line served as the control in this study.

To confirm the characteristics of primary cultured chicken TECs, reverse transcription-polymerase chain reaction (RT-PCR) analysis of epithelial cell-specific gene makers was performed. Primers designed using NCBI Primer-BLAST ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/, Table 1) were used to confirm the expression of the epithelial cell-specific gene markers, namely retinoic acid responder, fibroblast growth factor (FGF)-binding protein, and virus-activating protease (VAP). The DF-1 cell line was used as the control and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as the housekeeping gene. Total RNA was extracted from chicken TECs, cDNA was synthesized, and RT-PCR was performed. The PCR product was electrophoresed on 2% agarose gel, stained using DyneStaningSTAR (Dyne Bio INC, Seongnam, Korea), and observed using a UV image analyzer.

Immunocytochemistry (ICC) analysis was performed to confirm the morphology and growth pattern of chicken TECs. To characterize epithelial cells, the expression of E-cadherin and zonula occludens (ZO)-1 in the epithelial cell membrane was confirmed. Chicken TECs were cultured in 24-well plates at 37°C. The cells were washed using 1× PBS, fixed for 10 min with 4% paraformaldehyde in 1× PBS, and permeabilized with 1× PBS containing 0.25% Triton X-100. Protein staining was performed using rabbit E-cadherin primary antibody (catalog# 139490, 1:100 dilution; US Biological, Salem, MA, USA) or rabbit ZO-1 polyclonal primary antibody (catalog# 61-7300, 1:50 dilution; Thermo Fisher Scientific, USA) followed by an Alexa Fluor 488 goat anti-rabbit immunoglobulin G (H+L) secondary antibody (catalog# A-11034, 1:1,000 dilution; Thermo Fisher Scientific, USA) incubation. After incubation with antibodies, the cells were rinsed and mounted with Fixed Cell ReadyProbes Reagent (DAPI, blue; Thermo Fisher Scientific, USA). Images were captured using the EVOS FLoid Cell Imaging Station (Life Technologies, Carlsbad, CA, USA).

The HPAIV A/duck/Vietnam/QB1207/2012 (H5N1), which we have previously characterized against several tissues [15,16], was used in this study.

After culturing the TECs, the medium was removed, and the cells were washed with 1× PBS. For virus infection, fresh culture medium containing the H5N1 AIV subtype at a multiplicity of infection (MOI) of 0.1 plaque-forming units (PFU)/cell was added. The cells were then incubated with the virus at 37°C for different durations (0, 3, 6, and 12 h), washed with 1× PBS, and harvested for further study.

Cultured primary TECs were rinsed twice with 1× PBS and treated with poly (I:C) (10 μM; #P1530, Sigma-Aldrich, USA) in the culture medium. The cells were then washed twice using 1× PBS and collected after 12h for gene expression validation. Specifically, the TECs were treated with various concentrations (0, 1, 5, and 10 μg/mL) of poly (I:C) for different durations (0, 12, and 24 h) to select an appropriate final concentration.

The cultured TECs were pretreated with BAY 11–7085 (5 μM, 3 h; Sigma-Aldrich, USA), SB202190 (10 μM, 1 h; InvivoGen, San Diego, CA, USA) and SP600125 (25 μM, 1 h; InvivoGen, USA) to inhibit the NF-κB and MAPK signaling pathways, respectively. The cells were then treated with poly (I:C) for 12 h and washed with 1× PBS as described above.

Cultured TECs were treated with H5N1 (MOI of 0.1 PFU/cell) or poly (I:C) and incubated at 37°C with 5% CO₂. The TECs were then collected, and RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, USA) by following the manufacturer’s instructions. For cDNA synthesis, 2 μg of total RNA was treated with 1.0 U DNase I (RNase-free) and used for cDNA synthesis with a RevertAid First Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA). The resulting cDNA was diluted to 100 ng/μL using nuclease-free water.

For the reverse transcription-quantitative polymersase chain reaction (RT-qPCR) analysis, Dyne qPCR 2x PreMIX (SYBR Green with low ROX; Dyne Bio INC, Korea) was used in a CFX connect Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. Primers designed with NCBI Primer-BLAST ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/, Table 2) were utilized. The reaction mixture (20 μL) comprised 10 μL of 2× SYBR Green Master Mix, 1 μL each of 10 pM forward and reverse primers, 1 μL of template cDNA, and 7.0 μL of nuclease-free water. The amplification steps were as follows: 5 min at 95°C, 40 cycles of 30 s at 94°C, 50°C to 65°C annealing for 30 s, and extension for 30 s at 72°C. All the experiments were performed independently in triplicate. mRNA expression levels were normalized to those of GAPDH.

Primary chicken TECs were lysed in 100 μL radio-immunoprecipitation assay (RIPA) buffer supplemented with phosphatase inhibitor and protease inhibitor cocktails (Thermo Fisher Scientific, USA). The cells were collected from the culture plates and transferred to 1.7-mL Eppendorf tubes. The cells were centrifuged at 12,000×g for 15 min at 4°C, and the supernatant was stored at −80°C. Rabbit E-cadherin primary antibody (catalog# 139490, 1:500 dilution; US Biological, USA) and rabbit ZO-1 polyclonal primary antibody (catalog# 61-7300, 1:200 dilution; Thermo Fisher Scientific, USA) were used. Horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse (Thermo Fisher Scientific, USA) secondary antibodies were used based on the primary antibodies. Protein bands were detected using the Western Lightning Plus-ECL substrate (Thermo Fisher Scientific, USA). Protein expression levels were normalized to those of GAPDH (AM4300; Thermo Fisher Scientific, USA).

All data were analyzed using IBM SPSS Statistics for Windows, version 26.0 (IBM Corp., Armonk, NY, USA). Each RT-qPCR experiment was replicated three times. Statistical comparisons were performed in triplicate for two-group comparisons using the student’s t-test. Pairwise comparisons were performed using one-way analysis of variance (ANOVA) followed by Duncan’s post-hoc test. The data were presented as means± standard error of the mean. A difference was considered statistically significant when p<0.05, p<0.01, and p<0.001.

Chicken TECs were successfully isolated from 18-day-old embryonic chickens. After cell attachment, primary TECs at different incubation periods were observed under the microscope at 100× and 200× (Figure 1A) magnification. The TECs grew as a monolayer of cells with chicken embryonic fibroblasts. Moreover, these cells showed polygonal cell morphology, which is characteristic of epithelial cells. The cultured primary TECs formed colony-like cell clusters and proliferated around the clusters. In this study, 2 to 3 days were required for cells to attach and grow to 70% to 80% confluence or more in primary culture.

Epithelial cell characteristics were observed in TECs by RT-PCR and ICC analyses. The characteristics of primary TECs were confirmed using epithelial cell gene markers, including retinoic acid responder, FGF-binding protein, and VAP. The expression of epithelial cell-specific marker genes was detected in the TECs but not in the DF-1 cells (Figure 1B). In contrast, GAPDH expression did not differ between the chicken TECs, and the DF-1 cell line (Figure 1B).

To further confirm the morphology and growth pattern of the TECs, ICC analysis was performed, focusing on the expression of an epithelial membrane marker (Figure 1C). As expected, the membrane marker, E-cadherin was detected in the cultured primary TECs but not in the chicken fibroblast DF-1 cell line. The growth patterns showed the same colony-type cell clusters as described above (Figure 1). These results indicate that epithelial cells isolated from the trachea have properties based on the expression of several known epithelial-specific marker genes and junction proteins in the epithelial cell membrane.

To investigate the cellular immune response to HPAIV infection, the TECs were infected with H5N1 virus (MOI of 0.1 PFU/cell) in the culture media. After H5N1 infection, the TECs were collected to validate gene expression using RT-qPCR (Figure 2). The evaluation focused on measuring mRNA expression of antiviral genes in response to HPAIV infection and showed a significant increase in antiviral gene expression in the TECs infected with the H5N1 subtype (Figure 2). Over time, TLR3 and myeloid differentiation primary response (MYD) 88 expression increased and reached a peak value of 15.74- and 3.42-fold, respectively at 12 h. STAT1 expression significantly increased by 6.08-fold, 3.5-fold, and 6.92-fold, respectively, at all times of culture. Interferon (IFN)-α and interferon regulatory factor (IRF) 7 expression peaked at 12 h of incubation by 17.57- and 39.44-fold, respectively. Chemokine (C-C motif) ligand (CCL) 4 and interleukin (IL)-8 expression peaked at 6 h of incubation by 47.98- and 27.85-fold, respectively. Moreover, IL-6 expression increased over time and peaked at 12 h by 2.91-fold. These data indicate that primary chicken TECs infected with HPAIV recognize pathogens, trigger antiviral response, and produce antiviral molecules. Thus, TECs can express several innate immune genes as well as pro-inflammatory cytokines and chemokines in response to pathogens, including AIV.

To investigate the impact of poly (I:C) on antiviral gene expression, the TECs were treated with various concentrations of poly (I:C). Except for the expression of IFN-α, that of antiviral genes and chemokines, such IFN-β, IL-1β, and CCL4 increased in a concentration-dependent manner (Figure 3A). Additionally, IFN-β, IL-1β, and CCL4 reached their peak expression levels 12 h after treatment with 10 μg/mL poly (I:C) (Figure 3B).

We also measured the expression of genes related to the TLR and JAK/STAT signaling pathways as well as that of pro-inflammatory cytokines and chemokines in TECs 12 h after treatment. Compared with the control cells, the TECs treated with poly (I:C) demonstrated TLR3 upregulation. TECs treated with poly (I:C) significantly induced the expression of transcription factors, IRF7 and NF-κB1 by 10.79- and 3.12-fold, respectively. Moreover, MYD88, the signaling adaptor for TLRs (except TLR3), was upregulated by 3.45-fold (Figure 4).

Treatment of the TECs with poly (I:C) on TECs highly induced the expression of IFN-α, IFN-β, and IFN-γ by 4.06-, 4.66-, and 21.85-fold, respectively. The expressions of JAK1, STAT1, and STAT2 were significantly upregulated by 2.02-, 14.09-, and 25.16-fold, respectively (Figure 4). Moreover, MX1 and 2′-5′-oligoadenylate synthetase-like protein (OASL) were significantly upregulated by 7.29-fold, and 2300-fold, respectively. Poly (I:C) treatment also caused upregulation of CCL4 and C-X-C chemokine receptor (CXC) R1 expression by 6.33- and 3.23-fold, respectively (Figure 4).

To validate the impact of the NF-κB and MAPK signaling pathways in TECs, pathway inhibitors were employed to suppress their activation. Compared with the control TECs, the ones treated with poly (I:C) exhibited significantly increased antiviral response, leading to a significantly high expression of IRF7, IL-6, and IFN-α inducible protein 6 (IFI6) (Figure 5).

Furthermore, the results demonstrated that treatment with the signaling pathway inhibitors, namely BAY 11–7085 (NF-κB inhibitor), SB202190 (p38 MAPK inhibitor), and SP600125 (c-Jun NH2-terminal kinase [JNK] MAPK inhibitor) effectively decreased the antiviral effects induced by poly (I:C) treatment. The expression of the IRF7, IL-6, and IFI6 genes was significantly lesser in the TECs treated with each signaling pathway inhibitor and poly (I:C) than that of those in the cells treated only with poly (I:C) (Figure 5).

The impact of poly (I:C) treatment on junction proteins in the TECs, which regulate the physical barrier function, was investigated. After 24 h of poly (I:C) stimulation, ICC and western blotting were performed to assess the expression patterns of junction proteins, specifically E-cadherin and ZO-1. ICC analysis revealed no significant difference between the control and poly (I:C)-treated TECs (Figure 6A). However, western blotting results demonstrated that poly (I:C) treatment led to decreased protein expression levels of E-cadherin in adherens junctions, while those of ZO-1 in tight junctions did not change significantly (Figure 6B). Similarly, the mRNA expression of E-cadherin decreased in the TECs after poly (I:C) treatment, whereas that of ZO-1 did not differ significantly (Figure 6C). The results indicate that TECs act as a physical barrier and poly (I:C) treatment results in the loss of epithelial cell junction proteins.