Figure 1.
mRNA expression of TLR3, TLR7, TLR9, RIG-I and MDA-5 in human nasal mucosa.
mRNA expression in nasal mucosal biopsies was determined by real-time reverse transcriptase-PCR (n = 20). Data is presented in relation to β-actin as 2-ΔCt × 105 and depicted in log scale as mean ± SEM.
Figure 2.
The nasal epithelium expresses TLR3, TLR7, TLR9, RIG-I and MDA-5.
Sections of nasal biopsies were incubated with antibodies against TLR3 (A), TLR7 (B), TLR9 (C), RIG-I (D), and MDA-5 (E) and visualized by 3, 3′-diaminobenzidine (brown). In control slides (F), N-series universal negative control reagent was used. All sections were accompanied with a square magnification. All slides were counterstained with haematoxylin (blue). The figure shows one representative biopsy out of four (3 male, 1 female). The arrows indicate positive stained cells.
Figure 3.
mRNA expression of TLR3, TLR7, TLR9, RIG-I and MDA-5 in epithelial cells.
Levels of innate immune receptors in human nasal epithelial cells (HNEC) (n = 5) (A), Detroit-562 (n = 6) (B), and FaDu (n = 4) (C) was determined by real-time reverse transcriptase-PCR. Data is presented in relation to β-actin as 2-ΔCt × 105 and depicted in linear scale as mean ± SEM.
Figure 4.
Expression of TLR3, TLR7, TLR9, RIG-I and MDA-5 on epithelial cells.
Epithelial cells from primary HNEC (A–E), Detroit-562 (G–K) and FaDu (M–Q) were incubated with antibodies against TLR3, TLR7, TLR9, RIG-I, and MDA-5 and visualized by 3, 3′-diaminobenzidine (brown). In controls, N-series universal negative control reagent was used (F, L, R). All cells were counterstained with haematoxylin (blue). The figure shows one representative staining out of three independent experiments. The markers in the figure are 50 µm.
Figure 5.
Expression of TLR3, TLR7, TLR9, RIG-I and MDA-5 proteins on epithelial cells.
HNEC (A), Detroit-562 (B) and FaDu (C) were stained intracellularly with Abs against TLR3, TLR7, TLR9, RIG-I and MDA-5 (open histograms) or appropriate isotype control (shaded histograms) and analyzed by flow cytometry. Representative pictures from one out of three independent experiments are shown.
Figure 6.
TLR3, TLR7, TLR9, RIG-I and MDA-5 stimulation promotes cytokine release.
Nasal biopsies and epithelial cells were cultured in the absence (Untreated) or presence of 10 µg/ml poly(I:C) (TLR3), 10 µg/ml R-837 (TLR7), 1 µM CpG (TLR9) and 1 µg/ml poly(I:C)/LyoVec (RIG-I/MDA-5). TNF-α (10 ng/ml) was used as a positive control (data not shown). After 24 h, supernatants from nasal biopsies (n = 5) (A–D), HNEC (n = 6–9) (E–H), Detroit-562 (n = 5) (I–L) and FaDu (n = 5–9) (M–P) were collected and analyzed for levels of IL-6, IL-8, GM-CSF and IFN-β using ELISA. Data is presented as mean ± SEM of 5 to 9 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.