Comparison of pro- and anti-inflammatory responses in paired human primary airway epithelial cells and alveolar macrophages

Abstract

Background: Airway epithelial cells and alveolar macrophages (AMs) are the first line of defense in the lung during infection. Toll-like receptor (TLR) agonists have been extensively used to define the regulation of inflammation in these cells. However, previous studies were performed in non-paired airway epithelial cells and AMs. The major goal of our study was to compare the pro- and anti-inflammatory responses of paired human primary airway epithelial cells and AMs to TLR3 and TLR4 agonists.

Methods: Tracheobronchial epithelial cells (TBEC) and AMs from four smokers and four non-smokers without lung disease were cultured with or without Poly(I:C) (PIC) (a TLR3 agonist) or LPS (a TLR4 agonist) for 4, 24 and 48 h. The immune responses of paired cells were compared.

Results: TBEC and AMs showed stronger pro-inflammatory cytokine (e.g., IL-8) responses to PIC and LPS, respectively. TLR3 and TLR4 mRNA levels were similar in non-stimulated TBEC and AMs. However, PIC stimulation in AMs led to sustained up-regulation of the immune negative regulators Tollip and A20, which may render AMs less sensitive to PIC stimulation than TBEC. Unlike AMs, TBEC did not increase NF-κB activation after LPS stimulation. Interestingly, smoking status was correlated with less TLR3 and IRAK-M expression in non-stimulated TBEC, but not in AMs. PIC-stimulated TBEC and LPS-stimulated AMs from smokers vs. non-smokers produced more IL-8. Finally, we show that expression of A20 and IRAK-M is strongly correlated in the two paired cell types.

Conclusions: By using paired airway epithelial cells and AMs, this study reveals how these two critical types of lung cells respond to viral and bacterial pathogen associated molecular patterns, and provides rationale for modulating immune negative regulators to prevent excessive lung inflammation during respiratory infection.

Keywords: Alveolar macrophages; Cigarette smoke; Immune negative regulators; Inflammation; Pathogen associated molecular patterns; Toll-like receptor; Tracheobronchial epithelial cells.

Fig. 1

Characterization of primary human tracheobronchial epithelial cell (TBEC) and alveolar macrophages (AM) under submerged cultures. a TBEC showing features of basal cells with positive staining for cytokeratin-5 (KRT5 in red, DAPI in blue, 400X.); b Negative control for KRT5 stained with DAPI in blue (400X); c Primary alveolar macrophages (AM) stained positive for CD68 (red) and DAPI (blue, 400X); d Negative control for CD68, stained with DAPI in blue (400X)

Fig. 2

Human tracheobronchial epithelial cells (TBEC) and alveolar macrophages display different responses to different pathogen associated molecular patterns (PAMPs). IL-8 and IP-10 protein production in supernatants of cultured human TBEC (a, b and c) and cultured alveolar macrophages (d, e and f) in the absence (−) and presence of Poly(I:C) (PIC) or LPS treatment for 4, 24 and 48 h. N = 8 donor subjects

Fig. 3

PAMP stimulation increases inflammatory cytokine mRNA levels in primary human lung cells. mRNA expression of IL-8 (a) and IP-10 (b) in human tracheobronchial epithelial cells (TBEC) and alveolar macrophages in the absence (−) and presence of LPS and Poly(I:C) (PIC) at 24 h. N = 8 donor subjects. The horizontal lines indicate the medians

Fig. 4

Differential activation of NF-κB in human tracheobronchial epithelial cells (TBEC) and alveolar macrophages in responses to PAMPs. NF-κB p65 levels were measured in nuclear extracts from paired TBEC (a) and alveolar macrophages (b) in the absence (−) and presence of treatments with LPS and Poly(I:C) (PIC) at 4, 24 and 48 h. N = 3 donor subjects

Fig. 5

PAMP stimulation induces expression of negative regulators of TLR signaling in human tracheobronchial epithelial cells (TBEC). mRNA expression of negative regulators Tollip (a), A20 (b) and IRAK-M (c) in TBEC in the absence (−) and presence of LPS or Poly(I:C) (PIC) at 4, 24 and 48 h. N = 8 donor subjects. Median values are shown as horizontal lines. Densitometric analysis of Tollip (d) and A20 (e) western blots on lysates of TBEC in the absence (−) or presence of treatments with LPS, PIC at 4, 24 and 48 h. β-actin was included as a protein loading control to normalize Tollip or A20 expression. N = 3 donor subjects (additional blots in Additional file 1: Figure S2)

Fig. 6

PAMP stimulation induces expression of negative regulators of TLR signaling in alveolar macrophages. mRNA expression of the negative regulators Tollip (a), A20 (b) and IRAK-M (c) in macrophages in the absence (−) and presence of LPS, Poly(I:C) (PIC) at 4, 24 and 48 h. N = 8 donor subjects. Median values are shown as horizontal lines. Densitometric analysis of Tollip (d) and A20 (e) western blots on lysates of macrophages in the absence (−) or presence of treatments with LPS, PIC at 4, 24 and 48 h. β-actin was included as a protein loading control to normalize Tollip or A20 expression. N = 3 donor subjects (additional blots in Additional file 1: Figure S2)

Fig. 7

Expression of the negative regulators A20 and IRAK-M are strongly correlated between paired human tracheobronchial epithelial cells (TBEC) and alveolar macrophage samples from the same individual donors. A20 and IRAK-M mRNA expression showed significant correlation between the two cell types after 24 h of PIC stimulation. N = 8 donor subjects

Fig. 8

Smoking history alters IL-8 expressionat the protein level in human tracheobronchial epithelial cells (TBEC) and macrophages, and decreases TNF-α production in macrophages treated with LPS. a IL-8 production was measured in TBEC and alveolar macrophages in the absence (−) or presence of LPS or poly(I:C) (PIC) at 24 h, and compared between smokers (S, n = 4) and non-smokers (NS, n = 4). These data are a re-analysis of the data displayed in Fig. 3a. There is significant induction of IL-8 in smokers’ TBEC after PIC stimulation, and in smokers’ macrophages after LPS stimulation. b TNF-α production in supernatants of cultured alveolar macrophages. The cells from smokers (S, n = 4) and non-smokers (NS, n = 4) were treated in the absence (−) or presence of LPS or PIC for 4, 24 and 48 h. NS trend to have higher levels of TNF-α at all the time points (P = 0.1)

Fig. 9

Smoking history alters TLR expression in human tracheobronchial epithelial cells (TBEC) but not macrophages. TLR3 and TLR4 mRNA expression in TBEC (a and b) and alveolar macrophages (c and d) that were not treated, or treated with LPS or Poly(I:C) (PIC) for 4 and 24 h. N = 8 donor subjects including four smokers (S) and four non-smokers (NS)

Fig. 10

Proposed immune regulatory mechanisms of paired airway epithelial cells and lung macrophages in responses to Poly(I:C) and LPS stimulation