Table 1.
Epithelial gene specific primers.
Table 2.
Gene specific primers for RT-qPCR.
Figure 1.
Primary chicken tracheal epithelial cell culture (TEC).
Primary chicken tracheal epithelial cells were isolated and cultured as described in the Methods section. 1A: Primary chicken tracheal epithelial cells at 100X magnification. 1B: Confirmation of tracheal epithelial cell identity both in vitro and freshly isolated (ex vivo) from tracheae after ex-vivo exposure: PCR amplified epithelial cell specific genes from cDNA in agarose gel, compared to chicken embryonic fibroblast (DF-1) cells. 1C: Tracheal epithelial cells stained for E-cadherin and Vimentin at (400X magnification). Left panel shows TECs at different filter setting Blue (DAPI) for nuclear staining, Green (FITC) for Vimentin and Red (AlexaFluor 546) for E-cadherin, right panel shows merged picture for all filters. 1D: DF-1 fibroblast cells stained for E-cadherin and Vimentin at 400X magnification. Left panel shows DF-1 cells at different filter setting; Blue (DAPI) for nuclear staining, Green (FITC) for Vimentin and Red (AlexaFluor 546) for E-cadherin; right panel shows merged picture for all filters.
Figure 2.
Distribution of differentially regulated genes in TECs.
Differentially regulated genes (≥5 fold) in tracheal epithelial cell after exposure to live Rlow, Rhigh or LAMPs isolated from either strain 1.5 hours after exposure. The star (*) in the figure represent commonly up-regulated genes upon all four exposures, from which six follow up genes were chosen. n = 8 (4 biological replicates x2 dye swap technical replicates) for all microarray experiments.
Table 3.
Genes of significant interest from microarray analysis.
Table 4.
Pathway analysis of differentially regulated genes.
Figure 3.
Differential gene expression in TECs post-exposure.
mRNA fold difference in TECs exposed to Rlow, Rlow LAMP, Rhigh or Rhigh LAMP at 1.5, 6 and 24 hours respectively. Samples normalized to housekeeping gene GAPDH and un-exposed TECs as control. n = 6 for all experiments. Results are denoted as fold change ± SEM with all control values set at 1. Significant differences denoted as * = P<0.05, ** = P<0.01, *** = P<0.001. A: IL-12p40 mRNA. B: IL-8 mRNA. C: IL-6 mRNA. D: CCL-20 mRNA. E: NOS-2 mRNA. F: IL-1β mRNA.
Figure 4.
Differential gene expression in TECs exposed to LAMPs in the presence of signaling inhibitors.
Epithelial cells were exposed to LAMPs isolated from Rlow or Rhigh in the presence or absence of signaling inhibitors for 6 hours. Samples were normalized to the housekeeping gene GAPDH and un-exposed TECs served as control. n = 6 for all experiments. Results are denoted as fold change ± SEM with all control values set at 1. Significant differences denoted as * = P<0.05, ** = P<0.01, *** = P<0.001. A. IL-12p40. B. IL-1β. C. IL-8. D. IL-6. E. CCL-20. F. NOS-2.
Figure 5.
Differential gene expression in tracheal epithelial cells after ex-vivo exposure to LAMPs.
Comparison of mRNA fold difference in tracheal epithelial cells from tracheal explant exposed to Rlow, Rlow LAMP, Rhigh or Rhigh LAMP at 1.5 and 6 hours respectively. Samples normalized to housekeeping gene GAPDH and un-exposed tracheae as control. n = 6 for all experiments. Results are denoted as fold change ± SEM with all control values set at 1. Significant differences denoted as * = P<0.05, ** = P<0.01, *** = P<0.001. A: mRNA fold difference of all genes at 1.5 hours. B: mRNA fold difference of all genes at 6 hours.
Figure 6.
RT-qPCR analysis of miRNA and IL-10 differential expression in TECs.
Epithelial cells were exposed to Rlow, Rlow LAMP, Rhigh or Rhigh LAMP at 1.5, 6 and 24 hours respectively. Samples were normalized to housekeeping gene GAPDH and un-exposed TECs as control. n = 6 for all experiments. Results are denoted as fold change ± SEM with all control values set at 1. Significant differences denoted as * = P<0.05, ** = P<0.01, *** = P<0.001. A: mRNA fold difference of IL-10 in TECs at all three time points post exposure. B: mRNA fold difference of miRNA-146a in TECs at all three time points post exposure.