Comparison of innate immune agonists for induction of tracheal antimicrobial peptide gene expression in tracheal epithelial cells of cattle

Abstract

Bovine respiratory disease is a complex of bacterial and viral infections of economic and welfare importance to the beef industry. Although tracheal antimicrobial peptide (TAP) has microbicidal activity against bacterial pathogens causing bovine respiratory disease, risk factors for bovine respiratory disease including BVDV and stress (glucocorticoids) have been shown to inhibit the induced expression of this gene. Lipopolysaccharide is known to stimulate TAP gene expression, but the maximum effect is only observed after 16 h of stimulation. The present study investigated other agonists of TAP gene expression in primary cultures of bovine tracheal epithelial cells. PCR analysis of unstimulated tracheal epithelial cells, tracheal tissue and lung tissue each showed mRNA expression for Toll-like receptors (TLRs) 1-10. Quantitative RT-PCR analysis showed that Pam3CSK4 (an agonist of TLR1/2) and interleukin (IL)-17A significantly induced TAP gene expression in tracheal epithelial cells after only 4-8 h of stimulation. Flagellin (a TLR5 agonist), lipopolysaccharide and interferon-α also had stimulatory effects, but little or no response was found with class B CpG ODN 2007 (TLR9 agonist) or lipoteichoic acid (TLR2 agonist). The use of combined agonists had little or no enhancing effect above that of single agonists. Thus, Pam3CSK4, IL-17A and lipopolysaccharide rapidly and significantly induce TAP gene expression, suggesting that these stimulatory pathways may be of value for enhancing innate immunity in feedlot cattle at times of susceptibility to disease.

Figure 1

Histologic appearance of trypsinized primary cell cultures of bovine tracheal epithelial cells. A) Hematoxylin and eosin stain. B) Immunohistochemistry for pancytokeratin. C) Immunohistochemistry for vimentin, with immunolabeled cells shown by arrows.

Figure 2

Expression of Toll-like receptor (TLR) in tracheal epithelial cells, tracheal mucosa, and lung. Basal expression of mRNA expression for TLRs 1–10 was evaluated in cultured bovine tracheal epithelial cells, tracheal mucosal tissue, lung tissue, and positive control tissue (pooled mesenteric lymph node and ileum). Lanes 1–10: Samples of extracted RNA were reverse transcribed then amplified by PCR using specific primers, and the product was examined on an agarose gel. The gel lanes match the corresponding TLR (eg. the lane labelled 1 is TLR1), and the data shown from 1 calf are representative of the 3 calves examined. L: DNA molecular size standards: 400, 300, 200 and 100 bp.

Figure 3

Effects of Pam3CSK4, FSL-1, lipoteichoic acid and flagellin on tracheal antimicrobial peptide gene expression. Dose- and time-dependent effects were measured for Pam3CSK4 (A,B), a TLR2/1 agonist; FSL-1 (C,D), a TLR2/6 agonist; lipoteichoic acid (LTA) (E,F), a TLR2/2 agonist); and flagellin (G,H), a TLR5 agonist. For the dose–response studies (A,C,E,G), confluent bTEC were stimulated in triplicate with various concentrations of agonist for 16 h. For the time-course studies (B,D,F,H), confluent bTEC were stimulated in triplicate for 4, 8, 16 or 24 h with the doses of agonist shown. Gene expression of TAP relative to that of GAPDH was measured using real-time RT-qPCR. Lipopolysaccharide (LPS, 0.1 μg/mL) was used in all assays as a positive control and standard. *, significantly different from unstimulated cells (P < 0.05).

Figure 4

Effects of CpG oligodinucleotide, interleukin-17A and interferon-α on tracheal antimicrobial peptide gene expression. Dose- and time-dependent effects were measured for CpG oligodinucleotide (A,B), a TLR9 agonist; interleukin-17A (C,D); and interferon-α (E,F). For the dose–response studies (A,C,E), confluent bTEC were stimulated in triplicate with various concentrations of agonist for 16 h. For the time-course studies (B,D,F), confluent bTEC were stimulated in triplicate for 4, 8, 16 or 24 h with the doses of agonist shown. Gene expression of TAP relative to that of GAPDH was measured using real-time RT-qPCR. Lipopolysaccharide (LPS, 0.1 μg/mL) was used in all assays as a positive control and standard. *, significantly different from unstimulated cells (P < 0.05).

Figure 5

Comparison of the effects of IL-17A, Pam3CSK4 and LPS on tracheal antimicrobial peptide gene expression. Confluent cultures of tracheal epithelial cells from 4 different calves were non-stimulated (NS) or stimulated with 1 μg/mL Pam3CSK4, 316 ng/mL IL-17A, or 0.1 μg/mL LPS for 8 h (A) and 16 h (B) in triplicate. Gene expression was assessed using real-time RT-qPCR. Pam3CSK4 induced significantly higher tracheal antimicrobial peptide gene expression than IL-17A and LPS at both 8 and 16 h (P < 0.05). IL-17A induced significantly higher gene expression than LPS at 8 h (P < 0.05).

Figure 6

Effect of stimulation with single agonists compared to combined agonists. Cultured bovine tracheal epithelial cells were stimulated for 16 h in triplicate with various combinations of 1 μg/mL Pam3CSK4, 316 ng/mL IL-17A, or 0.1 μg/mL LPS. Tracheal antimicrobial peptide gene expression was measured as above. The effects of combined agonists were greater than that of interleukin-17A (IL-17A) alone, but minimally or not different than that of lipopolysaccharide (LPS) or Pam3CSK4 alone. The data shown (panels A, B and C) represent 3 studies conducted on different days using cells from different calves.