Local Innate Responses to TLR Ligands in the Chicken Trachea

Abstract

The chicken upper respiratory tract is the portal of entry for respiratory pathogens, such as avian influenza virus (AIV). The presence of microorganisms is sensed by pathogen recognition receptors (such as Toll-like receptors (TLRs)) of the innate immune defenses. Innate responses are essential for subsequent induction of potent adaptive immune responses, but little information is available about innate antiviral responses of the chicken trachea. We hypothesized that TLR ligands induce innate antiviral responses in the chicken trachea. Tracheal organ cultures (TOC) were used to investigate localized innate responses to TLR ligands. Expression of candidate genes, which play a role in antiviral responses, was quantified. To confirm the antiviral responses of stimulated TOC, chicken macrophages were treated with supernatants from stimulated TOC, prior to infection with AIV. The results demonstrated that TLR ligands induced the expression of pro-inflammatory cytokines, type I interferons and interferon stimulated genes in the chicken trachea. In conclusion, TLR ligands induce functional antiviral responses in the chicken trachea, which may act against some pathogens, such as AIV.

Keywords: TLR ligands; chicken trachea; innate antiviral responses.

Figure 1

Relative expression of candidate genes interleukin 1 beta (IL-1β) (A); inducible nitric oxide synthase (iNOS) (B); interfon (IFN) regulatory factor 7 (IRF7) (C); IFN-α (D); IFN-β (E); oligoadenylate synthetase (OAS) (F); IFN induced transmembrane protein 5 (IFITM5) (G); protein kinase R (PKR) (H); Viperin (I), in the chicken trachea. Tracheal organ cultures (TOCs) were treated with two different concentrations of lipopolysaccharide (LPS) from Escherichia coli (E. coli) O26:B6 (1.0 µg/mL (LPS low) and 10 µg/mL (LPS high)), Pam3CSK4 (1.0 µg/mL (Pam3 low) and 10 µg/mL (Pam3 high)), CpG ODN 1826 (1.0 µg/mL (CpG ODN low) and 10 µg/mL (CpG ODN high)). Control groups received either non CpG ODN (10 µg/mL) or medium. Gene expression was assessed at 3, 8 and 18 h post-treatment using quantitative real-time (RT)-PCR, relative to the housekeeping gene β-actin. Gene expression is presented as fold change relative to the medium group in LPS and Pam3CSK4 (low and high doses) groups. Gene expression is presented as fold change relative to the non CpG ODN group in CpG (low and high doses) groups. Error bars represent standard errors of the means. Fold changes and standard errors were calculated using REST software. Significant up-regulation (p ≤ 0.05) is indicated by *. Significant down-regulation is indicated by ζ. There were six biological replicates in each group.

Figure 1

Relative expression of candidate genes interleukin 1 beta (IL-1β) (A); inducible nitric oxide synthase (iNOS) (B); interfon (IFN) regulatory factor 7 (IRF7) (C); IFN-α (D); IFN-β (E); oligoadenylate synthetase (OAS) (F); IFN induced transmembrane protein 5 (IFITM5) (G); protein kinase R (PKR) (H); Viperin (I), in the chicken trachea. Tracheal organ cultures (TOCs) were treated with two different concentrations of lipopolysaccharide (LPS) from Escherichia coli (E. coli) O26:B6 (1.0 µg/mL (LPS low) and 10 µg/mL (LPS high)), Pam3CSK4 (1.0 µg/mL (Pam3 low) and 10 µg/mL (Pam3 high)), CpG ODN 1826 (1.0 µg/mL (CpG ODN low) and 10 µg/mL (CpG ODN high)). Control groups received either non CpG ODN (10 µg/mL) or medium. Gene expression was assessed at 3, 8 and 18 h post-treatment using quantitative real-time (RT)-PCR, relative to the housekeeping gene β-actin. Gene expression is presented as fold change relative to the medium group in LPS and Pam3CSK4 (low and high doses) groups. Gene expression is presented as fold change relative to the non CpG ODN group in CpG (low and high doses) groups. Error bars represent standard errors of the means. Fold changes and standard errors were calculated using REST software. Significant up-regulation (p ≤ 0.05) is indicated by *. Significant down-regulation is indicated by ζ. There were six biological replicates in each group.

Figure 2

Relative expression of Toll-like receptor (TLR) 2 (A), 3 (B), 4 (C) and 21 (D) in the trachea. TOCs were treated with two concentrations of LPS from E. coli O26:B6 (1.0 µg/mL (LPS low) and 10 µg/mL (LPS high)), Pam3CSK4 (1.0 µg/mL (Pam3 low) and 10 µg/mL (Pam3 high)), CpG ODN 1826 (1.0 µg/mL (CpG ODN low) and 10 µg/mL (CpG ODN high)). Control groups received either non CpG ODN (10 µg/mL) or medium. Gene expression was assessed at 3, 8 and 18 h post-treatment using quantitative RT-PCR, relative to the housekeeping gene β-actin. Gene expression is presented as fold change relative to the medium group in LPS and Pam3CSK4 (low and high doses) groups. Gene expression is presented as fold change relative to the non CpG ODN group in CpG (low and high doses) groups. Error bars represent standard errors of the means. Fold changes and standard errors were calculated using REST software. Significant up-regulation (p ≤ 0.05) is indicated by *. Significant down-regulation is indicated by ζ. There were six biological replicates in each group.

Figure 3

Nitrite oxide (NO₂⁻) production in chicken macrophage cells (MQ-NCSU cell line). Chicken macrophages were stimulated with supernatants from TOCs (that were previously stimulated with TLR ligands). Nitrite oxide in MQ-NCSU supernatants was measured after 48 h of stimulation, via the Griess assay. Nitric oxide production in each group was compared to the cells without stimulation using a two-tailed Student’s t-test. Significant differences (p ≤ 0.05) between a test group and the group without receiving supernatant from TOC are indicated by *. SupTOC-24: culture supernatants from TOCs collected 24 h post-treatment with TLR ligands or medium. SupTOC-48: culture supernatants from TOCs collected 48 h post-treatment with TLR ligands or medium.

Figure 4

Supernatants from TOCs stimulated with TLR ligands reduced influenza virus replication in chicken macrophage cells. There were five groups. Three of them were treated with supernatants from TOCs treated with LPS from E. coli O26:B6, Pam3CSK4 and CpG ODN 1826 and one was treated with supernatant from TOCs that received only medium (untreated trachea), and the last group did not receive supernatant from TOCs. Cells were infected 6 h later with H4N6 avian influenza virus (MOI of 1.0). Virus titer (12 h after infection) was quantified by means of a TCID₅₀ assay. a–c columns with no common letter are significantly different (p ≤ 0.05). SupTOC-24h: culture supernatants from TOCs collected 24 h post-treatment with TLR ligands or medium. SupTOC-48h: culture supernatants from TOCs collected 48 h post-treatment with TLR ligands or medium.