Toll-like receptor expression and induction of type I and type III interferons in primary airway epithelial cells

Abstract

Interferons (IFNs) are a critical component of the first line of antiviral defense. The activation of Toll-like receptors (TLRs) expressed by dendritic cells triggers different signaling cascades that result in the production of large amounts of IFNs. However, the functional consequences of TLR activation and differential IFN production in specific cell populations other than antigen-presenting cells have not yet been fully elucidated. In this study, we investigated TLR expression and polarization in airway epithelial cells (AECs) and the consequences of TLR agonist stimulation for the production of type I (IFN-α/β) and type III (IFN-λ) IFNs. Our results show that the pattern of expression and polarization of all TLRs in primary AEC cultures mirrors that of the human airways ex vivo and is receptor specific. The antiviral TLRs (TLR3, TLR7, and TLR9) are mostly expressed on the apical cell surfaces of epithelial cells in the human trachea and in primary polarized AECs. Type III IFN is the predominant IFN produced by the airway epithelium, and TLR3 is the only TLR that mediates IFN production by AECs, while all TLR agonists tested are capable of inducing AEC activation and interleukin-8 production. In response to influenza virus infection, AECs can produce IFN-λ in an IFNAR- and STAT1-independent manner. Our results emphasize the importance of using primary well-differentiated AECs to study TLR and antiviral responses and provide further insight into the regulation of IFN production during the antiviral response of the lung epithelium.

Fig 1

TLR expression in the airway epithelium of the human trachea. Immunofluorescence with anti-human TLR-specific antibodies was used to detect the expression and distribution of TLR1 to -10 in AECs in tissue sections of human tracheas. The lumen of the airways is on the top.

Fig 2

TLR expression in human primary AEC cultures. Immunofluorescence with anti-human TLR-specific antibodies was used to detect the expression and distribution of TLR1 to -10 in polarized hAEC cultures. The apical surface is on the top.

Fig 3

Cell surface TLR expression in mouse epithelial cells and in Beas-2B cells. Flow cytometry was used to detect the cell surface expression of TLR1 to -10. (A) mTECs; (B) Beas-2B cells. The percent expression is shown in the top panels, and the MFI is shown in the bottom panels. Isotype control and secondary antibody staining were used as controls. The data are representative of two independent experiments (means and SD).

Fig 4

Kinetics of IFN production by human AECs in response to stimulation with PAMPs and influenza virus. (A) IFN-α; (B) IFN-β; (C) IFN-λ; (D) IL-8 at 24 h; (E) IL-6 at 24 h. Primary well-differentiated hAECs were incubated with Pam3CSK4, poly(I·C), LPS, RecFlast, CL075, or CpG or with 2 × 10⁵ PFU influenza virus H3N2, as indicated in Materials and Methods. Culture supernatants were harvested at 8, 16, and 24 h and analyzed by ELISA. The data are representative of two independent experiments with three samples per group (means and SD).

Fig 5

Type I IFN production by epithelial cells, dendritic cells, and alveolar macrophages. (A) mTECs; (B) Beas-2B cells; (C) bone marrow-derived mouse dendritic cells; (D) alveolar macrophage cell line MH-S. Cells were incubated with Pam3CSK4, poly(I·C), LPS, RecFlast, CL075, or CpG or with 2 × 10⁵ PFU influenza virus, as indicated in Materials and Methods. The levels of type I IFN bioactivity were measured in culture supernatants at 24 h. The data are representative of two independent experiments with three samples per group (means and SD).

Fig 6

Role of synergic TLR stimulation on the regulation of IFN-λ production in epithelial cells. Cells were stimulated with influenza virus, Pam3CSK4, poly(I·C), LPS, RecFlast, CL075, and CpG alone or in combination. The levels of IFN-λ and IL-8 were measured in culture supernatants at 24 h by ELISA. (A) IFN-λ production in Beas-2B cells; (B) IFN-λ production in primary hAECs; (C) IL-8 production in primary hAECs. The data are representative of two independent experiments with three samples per group (means and SD).

Fig 7

Regulation of IFN-β and IFN-λ production in mTECs. mTEC cultures were derived from wild-type, IFNAR^−/−, and Stat1^−/− mice. The cells were stimulated with poly(I·C) (left panels) and influenza virus (right panels) as described in Materials and Methods, and the concentrations of IFNs were measured by ELISA 24 h later. (A) IFN-λ in response to poly(I·C); (B) IFN-λ in response to influenza virus; (C) IFN-β in response to poly(I·C); (D) IFN-β in response to influenza virus. The data are representative of two independent experiments with three samples per group (means and SD). *, P ≤ 0.05; **, P ≤ 0.005.

Fig 8

TLR3-poly(I·C) interaction mediates IFN-λ production by AECs. (A) IFN-λ production in wild-type and TLR3^−/− mTECs in response to poly(I·C) stimulation. (B) Intracellular and cell surface TLR3 expression in primary hAECs. The left panel shows the percent expression, and the right panel shows the MFI. Cells were stained with anti-TLR3 or with rat IgG2a isotype control antibodies. (C) Epithelial IFN-λ production in the presence of anti-TLR3 blocking antibodies. (D) Epithelial IFN-λ production in the presence of bafilomycin. IFN-λ was measured in culture supernatants by ELISA 24 h after stimulation. The data are representative of two independent experiments with 3 samples per group. Error bars indicate the SD. *, P ≤ 0.05; ***, P ≤ 0.0005.